U.S. patent number 9,599,622 [Application Number 14/402,358] was granted by the patent office on 2017-03-21 for fluorogenic ph-sensitive dyes and their methods of use (ii).
This patent grant is currently assigned to Life Technologies Corporation. The grantee listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Daniel Beacham, Kyle Gee, Shih-Jung Huang, Michael Janes, Aleksey Rukavishnikov, Upinder Singh, Wenjun Zhou.
United States Patent |
9,599,622 |
Gee , et al. |
March 21, 2017 |
Fluorogenic pH-sensitive dyes and their methods of use (II)
Abstract
Disclosed herein are compounds, compositions, methods and kits
for detecting pH in samples using pH-sensitive fluorescent dyes.
The compounds disclosed herein are novel xanthene-derivative dyes
comprising an aniline moiety with one or more electron donating
groups, which dyes are for detecting pH in samples either in vitro
or in vivo. Also described herein are processes for preparing said
dyes for use in the disclosed compositions, methods and kits.
Inventors: |
Gee; Kyle (Springfield, OR),
Singh; Upinder (Eugene, OR), Rukavishnikov; Aleksey
(Eugene, OR), Beacham; Daniel (Eugene, OR), Huang;
Shih-Jung (Eugene, OR), Janes; Michael (Eugene, OR),
Zhou; Wenjun (Eugene, OR) |
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
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Assignee: |
Life Technologies Corporation
(Carlsbad, CA)
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Family
ID: |
48014358 |
Appl.
No.: |
14/402,358 |
Filed: |
March 14, 2013 |
PCT
Filed: |
March 14, 2013 |
PCT No.: |
PCT/US2013/031637 |
371(c)(1),(2),(4) Date: |
November 20, 2014 |
PCT
Pub. No.: |
WO2013/180813 |
PCT
Pub. Date: |
December 05, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20150218379 A1 |
Aug 6, 2015 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61653333 |
May 30, 2012 |
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61653616 |
May 31, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09B
11/24 (20130101); G01N 33/582 (20130101); C07D
311/90 (20130101); G01N 33/5005 (20130101); G01N
33/84 (20130101); A61K 49/0041 (20130101) |
Current International
Class: |
C09B
11/24 (20060101); C07D 311/90 (20060101); G01N
33/84 (20060101); G01N 33/58 (20060101); G01N
33/50 (20060101); A61K 49/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO-02/36832 |
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May 2002 |
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WO |
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WO-2005/098437 |
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Oct 2005 |
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WO |
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WO2013180811 |
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Dec 2013 |
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WO |
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Other References
PCT/US2013/031535, , "International Preliminary Report on
Patentability and Written Opinion mailed on Dec. 2, 2014", Dec. 2,
2014, 9 Pages. cited by applicant .
PCT/US2013/031535, , "International Search Report and Written
Opinion", 2013, 13 pgs. cited by applicant .
PCT/US2013/031637, , "International Preliminary Report on
Patentability", Dec. 11, 2014, 7 pages. cited by applicant .
PCT/US2013/031637, , "International Search Report and Written
Opinion", 2013, 9 pgs. cited by applicant.
|
Primary Examiner: Yoo; Sun Jae
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 national stage application from
PCT/US2013/031637, filed Mar. 14, 2013, which claims the benefit of
priority to U.S. Provisional Application Ser. No. 61/653,333, filed
May 30, 2012 and 61/653,616, filed May 31, 2012, which are herein
incorporated by reference in their entirety.
Claims
What is claimed is:
1. A pH-sensitive fluorescent dye compound of structural formula
(I): ##STR00029## wherein R.sup.1 is alkoxy or thioalkyl; R.sup.2
and R.sup.6, which may be the same or different, are each
independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group; R.sup.3 is
--NR'R'', wherein R' and R'', which may be the same or different,
are each independently alkyl or substituted alkyl; R.sup.4 is
selected from the group consisting of alkyl and substituted alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance; R.sup.a is H, alkyl, or substituted alkyl;
R.sup.b is alkyl or substituted alkyl; and R.sup.7, R.sup.8,
R.sup.9, and R.sup.10, which may be the same or different, are each
independently alkenyl.
2. The compound according to claim 1, wherein R.sup.1 is alkoxy or
thioalkyl; R.sup.2 and R.sup.6, which may be the same or different,
are each independently H or halogen; R.sup.3 is --NR'R'', wherein
R' and R'', which may be the same or different, are each
independently alkyl; R.sup.4 is alkyl; R.sup.5 is selected from the
group consisting of alkyl; substituted alkyl; alkenyl; substituted
alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl;
substituted heteroaryl; heterocyclyl; substituted heterocyclyl;
alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl;
alkylaryloxycarbonyl; alkylheteroaryl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c, wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c; and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkenyl.
3. The compound according to claim 1, wherein R.sup.1 is alkoxy or
thioalkyl; R.sup.2 and R.sup.6, which may be the same or different,
are each independently H, Cl or F; R.sup.3 is --NR'R'', wherein R'
and R'', which may be the same or different, are each independently
alkyl; R.sup.4 is alkyl; R.sup.5 is selected from the group
consisting of alkyl; substituted alkyl; alkenyl; substituted
alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl;
substituted heteroaryl; heterocyclyl; substituted heterocyclyl;
alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl;
alkylaryloxycarbonyl; alkylheteroaryl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c, wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c; and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkenyl.
4. The compound according to claim 1, wherein R.sup.1 is alkoxy or
thioalkyl; R.sup.2 and R.sup.6 are each H; R.sup.3 is --NR'R'',
wherein R' and R'', which may be the same or different, are each
independently alkyl; R.sup.4 is alkyl; R.sup.5 is selected from the
group consisting of alkyl; substituted alkyl; alkenyl; substituted
alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl;
substituted heteroaryl; heterocyclyl; substituted heterocyclyl;
alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl;
alkylaryloxycarbonyl; alkylheteroaryl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c, wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c; and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkenyl.
5. The compound according to claim 1, wherein R.sup.1 is methoxy;
R.sup.2 and R.sup.6 are each H; R.sup.3 is --NR'R'', wherein R' and
R'', which may be the same or different, are each independently
methyl or ethyl; R.sup.4 is methyl or ethyl; R.sup.5 is methyl;
ethyl; carboxyalkyl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c; and R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which
may be the same or different, are each independently propenyl.
6. A pH-sensitive fluorescent dye compound of structural formula
(II): ##STR00030## wherein R.sup.1 is alkoxy or thioalkyl; R.sup.2
and R.sup.6, which may be the same or different, are each
independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group; R.sup.3 is
--NR'R'', wherein R' and R'', which may be the same or different,
are each independently alkyl or substituted alkyl; R.sup.4 is
selected from the group consisting of alkyl and substituted alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance; R.sup.a is H, alkyl, or substituted alkyl;
R.sup.b is alkyl or substituted alkyl; and R.sup.7 and R.sup.8,
which may be the same or different, are each independently alkyl or
alkenyl.
7. The compound according to claim 6, wherein R.sup.1 is alkoxy or
thioalkyl; R.sup.2 and R.sup.6, which may be the same or different,
are each independently H or halogen; R.sup.3 is --NR'R'', wherein
R' and R'', which may be the same or different, are each
independently alkyl; R.sup.4 is alkyl; R.sup.5 is selected from the
group consisting of alkyl; substituted alkyl; alkenyl; substituted
alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl;
substituted heteroaryl; heterocyclyl; substituted heterocyclyl;
alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl;
alkylaryloxycarbonyl; alkylheteroaryl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c, wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c; and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
8. The compound according to claim 6, wherein R.sup.1 is alkoxy or
thioalkyl; R.sup.2 and R.sup.6, which may be the same or different,
are each independently H, Cl or F; R.sup.3 is --NR'R'', wherein R'
and R'', which may be the same or different, are each independently
alkyl; R.sup.4 is alkyl; R.sup.5 is selected from the group
consisting of alkyl; substituted alkyl; alkenyl; substituted
alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl;
substituted heteroaryl; heterocyclyl; substituted heterocyclyl;
alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl;
alkylaryloxycarbonyl; alkylheteroaryl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c, wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c; and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
9. The compound according to claim 6, wherein R.sup.1 is alkoxy or
thioalkyl; R.sup.2 and R.sup.6 are each H; R.sup.3 is --NR'R'',
wherein R' and R'', which may be the same or different, are each
independently alkyl; R.sup.4 is alkyl; R.sup.5 is selected from the
group consisting of alkyl; substituted alkyl; alkenyl; substituted
alkenyl; acyl; aryl; substituted aryl; carboxyalkyl; heteroaryl;
substituted heteroaryl; heterocyclyl; substituted heterocyclyl;
alkylcarboxy; alkylalkoxycarbonyl; alkylaminocarbonyl;
alkylaryloxycarbonyl; alkylheteroaryl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c, wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c; and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
10. The compound according to claim 6, wherein R.sup.1 is methoxy;
R.sup.2 and R.sup.6 are each H; R.sup.3 is --NR'R'', wherein R' and
R'', which may be the same or different, are each independently
methyl or ethyl; R.sup.4 is methyl or ethyl; R.sup.5 is methyl;
ethyl; carboxyalkyl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c; and R.sup.7 and R.sup.8, which may be the same or
different, are each independently methyl or propenyl.
11. A pH-sensitive dye compound selected from the group consisting
of: ##STR00031##
12. A composition for determining the pH of a sample, the
composition comprising: a) one or more of the pH-sensitive
fluorescent dye compounds according to claim 1; and b) a carrier,
wherein the one or more pH-sensitive fluorescent dye compounds are
present in an amount effective to detect the pH of the sample.
13. A composition for determining the pH of a sample, the
composition comprising: (a) one or more of the pH-sensitive
fluorescent dye compounds according to claim 1; and (b) an analyte,
wherein the one or more pH-sensitive fluorescent dye compounds are
present in an amount effective to detect the pH of the sample.
14. A method for determining the pH of a sample, the method
comprising: (a) contacting the sample with one or more of the
pH-sensitive fluorescent dye compounds according to claim 1 to form
a contacted sample; (b) incubating the contacted sample for an
appropriate amount of time to form an incubated sample; (c)
illuminating the incubated sample with an appropriate wavelength to
form an illuminated sample; and (d) detecting fluorescent emissions
from the illuminated sample; wherein the fluorescent emissions are
used to determine the pH of the sample.
15. A method for monitoring the pH inside a live cell, the method
comprising: (a) contacting the cell with one or more of the
pH-sensitive fluorescent dye compounds according to claim 1 to form
a contacted cell; (b) incubating the contacted cell for a
sufficient amount of time for the dye compound or composition to
enter the cell to form a labeled cell; (c) illuminating the labeled
cell with an appropriate wavelength to form an illuminated cell;
and (d) detecting fluorescent emissions from the illuminated cell;
wherein the fluorescent emissions are used to monitor the pH inside
the cell.
16. A method for detecting phagocytosis of a carrier molecule in
solution, the method comprising: (a) conjugating the carrier
molecule to one or more of the pH-sensitive fluorescent dye
compounds according to claim 1 to form a carrier conjugate; (b)
contacting the carrier conjugate with a cell to form a contacted
cell; (c) incubating the contacted cell to form an incubated
solution; (d) illuminating the incubated solution to form an
illuminated solution; and (e) detecting fluorescent emissions from
the illuminated solution; wherein fluorescent emissions indicate
phagocytosis of the carrier molecule.
17. A method for detecting a pH related intracellular process, the
method comprising: (a) contacting one or more of the pH-sensitive
fluorescent dye compounds according to claim 1 with a cell to form
a contacted cell; (b) incubating the contacted cell to form an
incubated solution; (c) illuminating the incubated solution to form
an illuminated solution; and (d) detecting fluorescent emissions
from the illuminated solution; wherein increased fluorescent
emissions indicates activation of the intracellular process.
18. A method for identifying a target cell in a population of
cells, wherein the target cell is differentially labeled relative
to neighboring cells within the population, the method comprising:
(a) contacting one or more of the pH-sensitive dye compounds
according to claim 1 with the population of cells to form a
contacted cell population; (b) incubating the contacted cell
population for a period of time sufficient for the one or more of
the pH-sensitive dye compounds to enter the target cell, thereby
forming an incubated cell population; and (c) illuminating the
incubated cell population, wherein the target cell is identified by
a differential label relative to neighboring cells within the
population.
19. A method for diagnosing or detecting a disease in a subject,
the method comprising: (a) contacting a sample obtained from a
subject suspected of having the disease with one or more of the
pH-sensitive dye compounds according to claim 1 to form a contacted
sample; (b) incubating the contacted sample for an appropriate
amount of time to form an incubated sample; (c) illuminating the
incubated sample with an appropriate wavelength to form an
illuminated sample; and (d) detecting fluorescent emissions from
the illuminated sample; wherein the fluorescent emissions are used
to diagnose or detect the disease.
20. A kit for determining the pH of a sample comprising: (a) one or
more of the pH-sensitive dye compounds according to claim 1; (b)
one or more containers; and optionally (c) instructions for
determining the pH of the sample.
Description
FIELD
Novel pH-sensitive fluorescent dyes and assays for use in a variety
of applications including monitoring of intracellular processes are
disclosed.
BACKGROUND
pH-sensitive fluorescent dyes employed in biological research and
medical diagnostics belong to two groups, each distinguished by the
origin of fluorescent responses to changes in pH. The first group
includes compounds having fluorescence controlled by the ionization
of phenolic hydroxyl groups in a fluorophore. Examples include
fluorescein, carboxyfluorescein, Oregon Green.RTM., SNARF.RTM.,
SNAFL.RTM., and HPTS indicators.
U.S. Patent Publication No. 2006/0051874 describes fluorescein-like
structures incorporated into a fluorescent detector for monitoring
pH of the blood in bank storages. Because the degree of ionization
of these types of molecules increases upon lowering the acidity of
the environment, they become more fluorescent as pH increases.
Fluorescent pH sensors of the second group include an amino group
(aliphatic or aromatic) as an indicator moiety along with a
reporter fluorescent dye moiety. When such a molecule absorbs a
photon creating an excited electronic state, the electron of the
amino group's unshared pair transfers to the orbital vacated by
excitation. Such an electron transfer, referred to as Photoinduced
Electron Transfer (PET) prevents the excited molecule from emission
transition, thus the fluorescence of the dye is quenched.
Protonation of the amino group changes the nature and energy of the
pair's orbital and stops the PET. As a result, the fluorescent
reporter moiety responds to a pH change. Because protonation of the
amino group cancels the quenching, the PET-based sensors become
more fluorescent as pH decreases.
Examples of PET-based pH sensors include LysoSensor.TM. dyes, which
contain a dimethylamino group as an indicator moiety and
CypHer.RTM. 5E dye which has an indolenine indicator group. One
disadvantage of these sensors is that the working range is shifted
to the acidic side because of the low pKa of the indicator amino
group.
A family of rhodamine-based pH sensors is described in PCT
Publication No. WO 2005/098437 (Smith et al.). The dyes have a
benzene ring substituted ortho to the xanthene moiety by --OH or
--SH (or their deprotonated forms), such that deprotonation to a
negatively charged state quenches the fluorescence and it is only
upon protonation of the negatively charged --O.sup.- or --S.sup.-
to a neutral state that the fluorescence is restored. Typically,
the pH at which this occurs is less than pH 6. WO 2005/098437
purports that the ionized state of the --OH or --SH group is
responsible for the pH response of the dye and that the strong
electron withdrawing properties of the tetramethylrhodamine moiety
in the dyes significantly decreases the pKa of the indicator group,
thus shifting the sensors' working range toward highly acidic pH
values. However, this limits the applicability of the dyes
described in WO 2005/098437 at a physiological pH (e.g., pH 6-7),
especially in biological systems. An additional disadvantage of
these dyes is that their pKa is not tunable. Furthermore, these
compounds have been found by us to be unstable in solution.
Accordingly, there is a need for additional pH-sensitive
fluorescent dyes with improved properties, including in at least
some compounds the ability to detect pH changes in biological
systems. It is an object of the present invention to develop a
novel class of relatively stable fluorescent pH sensors that
fluoresce in the red portion of the UV/VIS spectrum, preferably
with a working range towards neutral and other biologically
relevant pH values that mitigate or remove the disadvantages of the
compounds known in art.
SUMMARY
Described herein are compounds, compositions, methods and kits for
detecting pH in samples using pH-sensitive fluorescent dyes, which
in one aspect, are characterized by the omission of a hydroxyl or
thiol group as required by WO 2005/098437. In another aspect, the
pH-sensitive fluorescent dyes as disclosed herein allow for the
detection of fluorescent responses to changes in pH in the red
portion of the UV/VIS spectrum. The present teachings provide a new
family of pH-sensitive red fluorescent dyes, having significant and
unexpected advantages over existing fluorescent pH-sensors in that
the presence of a dialkylamino group para to the alkoxy substituent
in the aniline moiety results in physiological pKa values and the
pKa's of the dyes provided herein are also tunable. Therefore, the
pH-sensitive fluorescent dyes provided herein may be modified to
suit a particular application or condition to be analyzed.
In certain embodiments, novel dye compounds are provided for use as
fluorescent pH sensors, the dye compounds having structural formula
(I):
##STR00001## wherein
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl or substituted alkyl;
R.sup.4 is selected from the group consisting of alkyl and
substituted alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x, is a reactive group; -L-R.sub.x; and -L-S.sub.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance;
R.sup.a is H, alkyl, or substituted alkyl;
R.sup.b is alkyl or substituted alkyl; and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.10 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H or halogen;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.10 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H, Cl or F;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.10 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6 are each H;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.10 are as follows:
R.sup.1 is methoxy;
R.sup.2 and R.sup.6 are each H;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently methyl or ethyl;
R.sup.4 is methyl or ethyl;
R.sup.5 is methyl; ethyl; carboxyalkyl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c; wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c; and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently methyl or propenyl.
In certain embodiments, novel dye compounds are provided for use as
fluorescent pH sensors, the dye compounds having structural formula
(II):
##STR00002## wherein
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl or substituted alkyl;
R.sup.4 is selected from the group consisting of alkyl and
substituted alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance, wherein L is a linker, R.sub.x is a
reactive group, and 5, is a conjugated substance;
R.sup.a is H, alkyl, or substituted alkyl;
R.sup.b is alkyl or substituted alkyl; and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.8 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H or halogen;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.8 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H, Cl or F;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.8 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6 are each H;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.8 are as follows:
R.sup.1 is methoxy;
R.sup.2 and R.sup.6 are each H;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently methyl or ethyl;
R.sup.4 is methyl or ethyl;
R.sup.5 is methyl; ethyl; carboxyalkyl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c; wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c;
R.sup.7 and R.sup.8, which may be the same or different, are each
independently methyl or propenyl.
In certain embodiments, compositions are provided for determining
the pH of a sample, the compositions comprising:
a) one or more of the pH-sensitive fluorescent dye compounds
described herein; and
b) a carrier,
wherein the one or more pH-sensitive fluorescent dye compounds are
present in an amount effective to detect the pH of the sample.
In certain embodiments, compositions are provided for determining
the pH of a sample, the compositions comprising:
(a) one or more of the pH-sensitive fluorescent dye compounds
described herein; and
(b) an analyte,
wherein the one or more pH-sensitive fluorescent dye compounds are
present in an amount effective to detect the pH of the sample.
In certain embodiments, methods are provided for determining the pH
of a sample, the methods comprising:
(a) contacting the sample with one or more of the pH-sensitive
fluorescent dye compounds described herein to form a contacted
sample;
(b) incubating the contacted sample for an appropriate amount of
time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated
sample;
wherein the fluorescent emissions are used to determine the pH of
the sample.
In certain embodiments, methods are provided for determining the pH
of a sample, the methods comprising:
(a) contacting the sample with one or more of the compositions
described herein to form a contacted sample;
(b) incubating the contacted sample for an appropriate amount of
time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated
sample;
wherein the fluorescent emissions are used to determine the pH of
the sample.
In certain embodiments, methods are provided for monitoring the pH
inside a live cell, the methods comprising:
(a) contacting the cell with one or more of the pH-sensitive
fluorescent dye compounds described herein to form a contacted
cell;
(b) incubating the contacted cell for a sufficient amount of time
for the one or more pH-sensitive fluorescent dye compounds to enter
the cell to form a labeled cell;
(c) illuminating the labeled cell with an appropriate wavelength to
form an illuminated cell; and
(d) detecting fluorescent emissions from the illuminated cell;
wherein the fluorescent emissions are used to monitor the pH inside
the cell.
In certain embodiments, methods are provided for monitoring the pH
inside a live cell, the methods comprising:
(a) contacting the cell with one or more of the compositions
described herein to form a contacted cell;
(b) incubating the contacted cell for a sufficient amount of time
for the one or more compositions to enter the cell to form a
labeled cell;
(c) illuminating the labeled cell with an appropriate wavelength to
form an illuminated cell; and
(d) detecting fluorescent emissions from the illuminated cell;
wherein the fluorescent emissions are used to monitor the pH inside
the cell.
In certain embodiments, methods are provided for detecting
phagocytosis of a carrier molecule in solution, the methods
comprising:
(a) conjugating the carrier molecule to one or more of the
pH-sensitive fluorescent dye compounds described herein to form a
carrier-dye conjugate;
(b) contacting the carrier-dye conjugate with a cell to form a
contacted cell;
(c) incubating the contacted cell to form an incubated
solution;
(d) illuminating the incubated solution to form an illuminated
solution; and
(e) detecting fluorescent emissions from the illuminated
solution;
wherein fluorescent emissions indicate phagocytosis of the carrier
molecule.
In certain embodiments, methods are provided for detecting
phagocytosis of a carrier molecule in solution, the methods
comprising:
(a) conjugating the carrier molecule to one or more of the
compositions described herein to form a carrier-dye conjugate;
(b) contacting the carrier-dye conjugate with a cell to form a
contacted cell;
(c) incubating the contacted cell to form an incubated
solution;
(d) illuminating the incubated solution to form an illuminated
solution; and
(e) detecting fluorescent emissions from the illuminated
solution;
wherein fluorescent emissions indicate phagocytosis of the carrier
molecule.
In certain embodiments, methods are provided for detecting a pH
related intracellular process, the methods comprising:
(a) contacting any one of the pH-sensitive fluorescent dye
compounds described herein with a cell to form a contacted
cell;
(b) incubating the contacted cell to form an incubated
solution;
(c) illuminating the incubated solution to form an illuminated
solution; and
(d) detecting fluorescent emissions from the illuminated
solution;
wherein increased fluorescent emissions indicates activation of the
intracellular process.
In certain embodiments, methods are provided for detecting a pH
related intracellular process, the methods comprising:
(a) contacting any one of the compositions described herein with a
cell to form a contacted cell;
(b) incubating the contacted cell to form an incubated
solution;
(c) illuminating the incubated solution to form an illuminated
solution; and
(d) detecting fluorescent emissions from the illuminated
solution;
wherein increased fluorescent emissions indicates activation of the
intracellular process.
In certain embodiments, methods are provided for identifying a
target cell in a population of cells, wherein the target cell is
differentially labeled relative to neighboring cells within the
population, the methods comprising;
(a) contacting one or more of the pH-sensitive dye compounds
disclosed herein with the population of cells to form a contacted
cell population;
(b) incubating the contacted cell population for a period of time
sufficient for the one or more of the pH-sensitive dye compounds to
enter the target cell, thereby forming an incubated cell
population; and
(c) illuminating the incubated cell population, wherein the target
cell is identified by a differential label relative to neighboring
cells within the population.
In certain embodiments, methods are provided for identifying a
target cell in a population of cells, wherein the target cell is
differentially labeled relative to neighboring cells within the
population, the methods comprising;
(a) contacting one or more of the compositions disclosed herein
with the population of cells to form a contacted cell
population;
(b) incubating the contacted cell population for a period of time
sufficient for the one or more of the compositions to enter the
target cell, thereby forming an incubated cell population; and
(c) illuminating the incubated cell population, wherein the target
cell is identified by a differential label relative to neighboring
cells within the population.
In certain embodiments, methods are provided for diagnosing or
detecting a disease in a subject, the method comprising:
(a) contacting a sample obtained from a subject suspected of having
the disease with one or more of the pH-sensitive dye compounds
disclosed herein to form a contacted sample;
(b) incubating the contacted sample for an appropriate amount of
time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated
sample;
wherein the fluorescent emissions are used to diagnose or detect
the disease.
In certain embodiments, methods are provided for diagnosing or
detecting a disease in a subject, the methods comprising:
(a) contacting a sample obtained from a subject suspected of having
the disease with one or more of the compositions disclosed herein
to form a contacted sample;
(b) incubating the contacted sample for an appropriate amount of
time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated
sample;
wherein the fluorescent emissions are used to diagnose or detect
the disease.
In certain embodiments, kits are provided for determining the pH of
a sample comprising:
(a) one or more of the pH-sensitive fluorescent dye compounds
described herein;
(b) one or more containers; and optionally
(c) instructions for determining the pH of the sample.
In certain embodiments, kits are provided for determining the pH of
a sample comprising:
(a) one or more of the compositions described herein;
(b) one or more containers; and optionally
(c) instructions for determining the pH of the sample.
In certain embodiments, processes are provided of synthesizing a
compound of structural formula (I):
##STR00003##
the process comprising: contacting a compound of structural formula
(III):
##STR00004## with a compound of structural formula (IV):
##STR00005## to form a compound of structural formula (I),
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are as previously
defined.
Other embodiments and illustrative aspects, features and advantages
of the present invention will become apparent from the following
detailed description. It should be understood, however, that the
detailed description and the specific examples, while indicating
preferred embodiments, are given by way of illustration only, since
various changes and modifications within the spirit and scope of
the present invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
Although the following figures depict various examples of the
invention, the invention is not limited to the examples depicted in
the figures.
FIG. 1 is a schematic representation of the pH values for
intracellular compartments and organelles.
FIG. 2 describes cellular uptake of pH-sensitive fluorescent dye
compounds. Panel A is a schematic representation of cellular uptake
of pH-sensitive fluorescent dye-conjugated compounds including
dye-conjugated bacteria for monitoring phagocytosis and
dye-conjugated particles for monitoring endocytosis, using the
compounds and methods according to embodiments disclosed herein.
Panel B is a fluorescence micrograph showing cells that have
internalized a pH-sensitive fluorescent dye compound according to
certain embodiments of the present teachings.
FIG. 3 graphically shows the correlation of pH with fluorescence
for Compound (1). Panel A shows the fluorescence intensity of
Compound (1) as a function of pH. Panel B shows the pKa of Compound
(1).
FIG. 4 shows internalization of EGF conjugated to Compound (4). The
left panel shows cells pretreated with EGF only and the right panel
shows cells treated with dye-conjugated EGF.
FIG. 5 shows internalization of Compound (4) in cells.
FIG. 6 shows the change of fluorescent signal from the Compound (4)
located inside the cell as the external pH changes that reflects
the equilibration of protons through the nigericin pores in the
cellular membrane.
FIG. 7 shows cycling of cytosolic pH as measured by fluorescence of
cells labeled with Compound (4).
FIG. 8 shows the pH activation of fluorescent signal from IgG
conjugates (IgG conjugated to Compound (1)).
DETAILED DESCRIPTION
A family of rhodamine-based pH sensors (Smith, et. al; PCT
Publication No. WO 2005/098437, herein incorporated by reference in
its entirety) displays pH-dependency based on the ionization state
of the X substituent (see Scheme I below), namely a hydroxyl or
thiol group, such that deprotonation of the --OH or --SH group to a
negatively charged state quenches the fluorescence. It is only upon
protonation of the negatively charged --O.sup.- or --S.sup.- that
the fluorescence is restored as illustrated in Scheme 1:
##STR00006##
However, we have unexpectedly found that alkoxy substitutions at
the corresponding X position are still capable of demonstrating a
modulated fluorescence in response to a change in pH. Thus, while
not wishing to be bound by a theory, we postulate that it is the
protonation of the nitrogen at the 4 position on the aryl ring that
modulates fluorescence. In any event, the present invention is
predicated on the surprising discovery that the hitherto
indispensable hydroxy or thiol group X may be dispensed with
provided that a substituent nitrogen is retained. Advantageously,
the addition of a dialkylamino group at the R.sup.3 position
resulted in a physiological pKa.
In addition, the pH-sensitive fluorescent dyes provided herein
fluoresce in the red portion of the UV/VIS spectrum and have
different chemical behavior as compared to other pH-sensitive dyes.
It was surprisingly discovered that: 1) the electron withdrawing
power of the aniline moiety may be modulated by adding various
electron donating groups to the benzene ring, and 2) the addition
of electron donating groups such as dialkylamino groups at the
R.sup.3 position and/or halogen at positions R.sup.2 and/or
R.sup.6, may be used to tune the pKa of the pH-sensitive
fluorescent dyes to be at or near physiological pH. Furthermore, by
altering the electron donating groups at positions R.sup.1-R.sup.6,
the pKa of the pH-sensitive fluorescent dye may be modulated to
suit a particular need.
Further, we have found that the dyes described by Smith et al.
(supra) are not stable in solution, most likely as a result of
aerobic oxidation (e.g., oxidation by exposure to ambient air). In
addition, strong electron withdrawing properties of the reporter
tetramethylrhodamine moiety of the dyes described in Smith et al.
significantly decrease the pKa of the indicator group at the X
position of Scheme 1, thus shifting the sensors' working range
towards acidic pH values.
In certain embodiments, therefore, the present invention provides
pH-sensitive fluorescent dyes having an aniline moiety (of which
the amino group may be substituted or modified as disclosed herein)
wherein the benzene ring of the aniline moiety is free from hydroxy
and thiol substituents ortho to the fluorophore or, in certain
embodiments, free from hydroxy and thiol at all positions. In
particular, these pH-sensitive fluorescent dye compounds may have,
in place of the hydroxy or thiol substituent required by Smith et
al. (supra), a moiety wherein the oxygen or sulfur of the hydroxy
or thiol group has been incorporated into an ether or thioether
linkage, for example as part of an alkoxy group or furan moiety, or
their sulfur analogs. Viewed alternatively, the pH-sensitive
fluorescent dye compounds provided herein which retain the oxygen
or sulfur in etherified form, are dye compounds which provide
increased electronic density of the molecule through strategic
introduction of electron donating groups (EDG) to the benzene ring
resulting in an electron rich aniline moiety, thereby moving the
pKa closer to a physiological range (e.g., pH 6-8). Accordingly,
the benzene ring may be substituted one or more times (e.g., 1, 2,
3 or 4 times) by an electron donating group, the electron donating
groups being the same or different. In certain embodiments, the
pH-sensitive fluorescent dye compounds provided herein have the
etherified O or S replaced by another electron donating group.
Irrespective of whether the etherified O or S is replaced by
another EDG, supplementary electron donating groups may be provided
on the benzene ring to further modulate the pKa. In certain
embodiments, the pH-sensitive fluorescent dye compounds provided
herein may comprise two electron donating groups in total on the
benzene ring, in particular two electron donating groups of the
type having a lone pair of electrons available immediately next to
the benzene ring (e.g. alkoxy or dialkylamino, optionally
substituted as described herein). Additionally, modifications may
be made to modulate the quantum yield of the pH-sensitive
fluorescent dye compounds of the present disclosure. Thus, the
pH-sensitive fluorescent dye compounds as disclosed herein have
significant advantages over other PET-based dyes and advantageously
provide the benefit of having improved stability and/or a pKa in
the range of physiological applications. In addition, the pKa of
the pH-sensitive fluorescent dye compounds provided herein may be
tuned to particular pKa's depending on the electron donating
group(s) used on the benzene ring (e.g., on the aniline
moiety).
In certain embodiments, the pKa of the aniline's amino group is
modulated by modifying the amino group into a more basic nitrogen
functional group. This feature may usefully be adopted as an
alternative to replacing the omitted hydroxy or thiol group with
another electron donating group; alternatively, modification of the
amino group into a more basic group may be combined with
substitution of the benzene ring by at least one electron donating
group other than a hydroxy or thiol group.
Also included herein are embodiments in which the pKa of the
aniline moiety is modulated by modifying the amino group at
position R.sup.3 to --NR'R'', wherein R' and R'', which may be the
same or different, are each independently alkyl or substituted
alkyl (e.g., a dialkylamino group) in order to bring the pKa closer
to the physiological range. In certain embodiments, the
dialkylamino group is dimethylamino (e.g., --N(CH.sub.3).sub.2). In
certain embodiments, the dialkylamino group is diethylamino (e.g.,
--N(CH.sub.2CH.sub.3).sub.2).
Particular features targeted by the compounds, compositions,
methods and kits described herein include one or more of: (1)
dissociation constant pKa within the physiological range; (2)
greater stability (considered to be towards oxidation); (3)
flexible synthetic methods allowing introduction of pKa-modulating
substituents along with reactive groups; and 4) tunability of the
pKa. As described previously herein, the pH-sensitive fluorescent
dye compounds of the present invention, have enhanced stability by
dispensing with the previously indispensable hydroxy or thiol at
position X of Scheme I, and the hydroxy or thiol group is
advantageously replaced by another electron donating group at the
same position, such as an alkoxy or thioalkyl, preferably an
alkoxy. Additionally or alternatively, such other electron donating
groups may be substituted at other positions on the benzene ring.
In certain embodiments, such other electron donating groups include
dialkylamino groups, wherein each alkyl group, which may be the
same or different, is each independently alkyl or substituted
alkyl.
In order to achieve these goals a novel class of the pH-sensitive
compounds was designed, synthesized and tested in analytical and
biological applications. The structures of the preferred dye
compounds include structural formulae (I) and (II).
Substituents on an aromatic ring may either donate electrons to the
aromatic ring or withdraw electrons from the aromatic ring as
compared to a hydrogen atom attached to the ring. Substituents may
therefore be classified as electron donating groups or electron
withdrawing groups.
Many electron donating groups have lone pairs of electrons on the
atom adjacent to the pi (.pi.) system of the aromatic ring. Alkyl,
aromatic and alkenyl groups are examples of electron donating
groups. Electron withdrawing groups are generally those where the
atom adjacent to the aromatic pi system has a formal positive
charge or a .delta. positive charge (for example, due to being
connected to more electronegative atoms). Electron donating groups
have an activating effect with respect to further substitution of
the ring system and tend to direct further substitution ortho/para,
while electron withdrawing groups are deactivating and tend to
direct further substitution meta. The exception to this is halogen
substituents, which, while overall electron withdrawing and
deactivating, tend to direct further substitution ortho/para due to
resonance (lone pair) donation. Table 1 indicates the relative
electron withdrawing and donating character of some common
substituents.
TABLE-US-00001 TABLE 1 Relative electron donating/withdrawing
character of different aromatic ring substituents, ranked from most
electron donating to most electron withdrawing Activating/
Substituent Character relative to H deactivating Directing
--O.sup.- electron donating strongly activate ortho/para --NR.sub.2
electron donating strongly activate ortho/para --NH.sub.2 electron
donating strongly activate ortho/para --OH electron donating
strongly activate ortho/para --OR electron donating strongly
activate ortho/para --NHC(O)R electron donating moderately activate
ortho/para --OC(O)R electron donating moderately activate
ortho/para --R electron donating weakly activate ortho/para --Ph
electron donating weakly activate ortho/para --CH.dbd.CR.sub.2
electron donating weakly activate ortho/para --H reference neutral
ortho/para --X (X = halo) electron withdrawing weakly deactivate
ortho/para --C(O)H electron withdrawing moderately meta deactivate
--C(O)R electron withdrawing moderately meta deactivate --C(O)OR
electron withdrawing moderately meta deactivate --C(O)OH electron
withdrawing moderately meta deactivate --CF.sub.3 electron
withdrawing strongly deactivate meta --CN electron withdrawing
strongly deactivate meta --S(O).sub.2OH electron withdrawing
strongly deactivate meta --N.sup.(+)H.sub.3 electron withdrawing
strongly deactivate meta --N.sup.(+)R.sub.3 electron withdrawing
strongly deactivate meta --N.sup.(+)(O)O.sup.(-) electron
withdrawing strongly deactivate meta
The symbol R in Table 1 in particular stands for alkyl, though it
may be substituted in any reasonable way which does not transform
the electronic effect of alkyl from donating to withdrawing or
vice-versa. This specification further describes suitable electron
donating groups for phenylic substitution of the aniline or
aniline-like ring described in the specification.
DEFINITIONS
To more clearly and concisely describe and point out the subject
matter of the present teachings, the following definitions are
provided for specific terms, which are used in the following
description and appended claims. Throughout the specification,
exemplification of specific terms should be considered as
non-limiting examples.
Before describing the present teachings in detail, it is to be
understood that the disclosure is not limited to specific
compositions or process steps, as such may vary. It should be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural references unless
the context clearly dictates otherwise. Thus, for example,
reference to "a fluorescent pH sensitive dye" includes a plurality
of dyes and reference to "a cell" includes a plurality of cells and
the like. The phrase "and/or" denotes a shorthand way of indicating
that the specific combination is contemplated in combination and
separately, in the alternative. For illustration purposes, but not
as a limitation, "X and/or Y" can mean "X" or "Y" or "X" and
"Y".
It will be appreciated that there is an implied "about" prior to
the temperatures, concentrations, times, etc. discussed in the
present disclosure, such that slight and insubstantial deviations
are within the scope of the present teachings herein. Also, the use
of "comprise", "comprises", "comprising", "contain", "contains",
"containing", "include", "includes", and "including" are not
intended to be limiting. It is to be understood that both the
foregoing general description and detailed description are
exemplary and explanatory only and are not restrictive of the
teachings.
Unless specifically noted in the above specification, embodiments
in the above specification that recite "comprising" various
components are also contemplated as "consisting of" or "consisting
essentially of" the recited components; embodiments in the
specification that recite "consisting of" various components are
also contemplated as "comprising" or "consisting essentially of"
the recited components; and embodiments in the specification that
recite "consisting essentially of" various components are also
contemplated as "consisting of" or "comprising" the recited
components (this interchangeability does not apply to the use of
these terms in the claims).
The term "or combinations thereof" as used herein refers to all
permutations and combinations of the listed terms preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, ACB,
CBA, BCA, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and
so forth. The skilled artisan will understand that typically there
is no limit on the number of items or terms in any combination,
unless otherwise apparent from the context.
The section headings used herein are for organizational purposes
only and are not to be construed as limiting the desired subject
matter in any way. All literature cited in the specification,
including but not limited to, patents, patent applications,
articles, books and treatises are expressly incorporated by
reference in their entirety for any purpose. In the event that any
of the incorporated literature contradicts any term defined in this
specification, this specification controls. While the present
teachings are described in conjunction with various embodiments, it
is not intended that the present teachings be limited to such
embodiments. On the contrary, the present teachings encompass
various alternatives, modifications, and equivalents, as will be
appreciated by those of skill in the art.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. The
following terms are defined for purposes of the teachings as
described herein.
"Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups
having from 1 to 10 carbon atoms and preferably 1 to 6 carbon
atoms, e.g. 1, 2, 3, 4, 5 or 6 carbon atoms. This term includes, by
way of example, linear and branched hydrocarbyl groups such as
methyl (CH.sub.3--), ethyl (CH.sub.3CH.sub.2--), n-propyl
(CH.sub.3CH.sub.2CH.sub.2--), isopropyl ((CH.sub.3).sub.2CH--),
n-butyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2--), isobutyl
((CH.sub.3).sub.2CHCH.sub.2--), sec-butyl
((CH.sub.3)(CH.sub.3CH.sub.2)CH--), t-butyl ((CH.sub.3).sub.3C--),
n-pentyl (CH.sub.3CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and
neopentyl ((CH.sub.3).sub.3CCH.sub.2--).
"Substituted alkyl" refers to an alkyl group having from 1 to 5,
preferably 1 to 3, or more preferably 1 to 2 substituents selected
from the group consisting of alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxylalkyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, cycloalkyl, substituted cycloalkyl,
cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,
substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyl,
cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio,
substituted cycloalkenylthio, guanidino, substituted guanidino,
halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy,
substituted heteroaryloxy, heteroarylthio, substituted
heteroarylthio, heterocyclic, substituted heterocyclic,
heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,
substituted heterocyclylthio, nitro, SO.sub.3H, substituted
sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, and substituted
alkylthio, wherein said substituents are defined herein. Particular
substituted alkyl groups comprise a reactive group for direct or
indirect linking to a carrier molecule or solid support; as
examples may be mentioned alkyl substituted by carboxyl or a
carboxyl ester (e.g. an activated ester such as an
N-hydroxysuccinimide ester) and alkyl substituted by aminocarbonyl
--CONHR where R is an organic moiety as defined below with
reference to the term "aminocarbonyl", e.g. a C.sub.1-C.sub.10
(e.g. C.sub.1-C.sub.6) alkyl terminally substituted by a reactive
group (R.sub.x) including, but not limited to, carboxyl,
carboxylester, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, and DIBO-amine.
"Alkoxy" refers to the group --O-alkyl wherein alkyl is defined
herein. Alkoxy includes, by way of example, methoxy, ethoxy,
n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and
n-pentoxy.
"Substituted alkoxy" refers to the group --O-(substituted alkyl),
wherein substituted alkyl is defined herein.
"Acyl" refers to the groups H--C(O)--, alkyl-C(O)--, substituted
alkyl-C(O)--, alkenyl-C(O)--, substituted alkenyl-C(O)--,
alkynyl-C(O)--, substituted alkynyl-C(O)--, cycloalkyl-C(O)--,
substituted cycloalkyl-C(O)--, cycloalkenyl-C(O)--, substituted
cycloalkenyl-C(O)--, aryl-C(O)--, substituted aryl-C(O)--,
heteroaryl-C(O)--, substituted heteroaryl-C(O)--,
heterocyclic-C(O)--, and substituted heterocyclic-C(O)--, wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein. Acyl includes the "acetyl"
group CH.sub.3C(O)--.
"Acylamino" refers to the groups NRC(O)alkyl, --NRC(O)substituted
alkyl, --NRC(O)cycloalkyl, --NRC(O)substituted cycloalkyl,
--NRC(O)cycloalkenyl, --NRC(O)substituted cycloalkenyl,
--NRC(O)alkenyl, --NRC(O)substituted alkenyl, --NRC(O)alkynyl,
--NRC(O)substituted alkynyl, --NRC(O)aryl, --NRC(O)substituted
aryl, --NRC(O)heteroaryl, --NRC(O)substituted heteroaryl,
--NRC(O)heterocyclic, and --NRC(O)substituted heterocyclic, wherein
R is hydrogen or alkyl and wherein alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Acyloxy" refers to the groups alkyl-C(O)O--, substituted
alkyl-C(O)O--, alkenyl-C(O)O--, substituted alkenyl-C(O)O--,
alkynyl-C(O)O--, substituted alkynyl-C(O)O--, aryl-C(O)O--,
substituted aryl-C(O)O--, cycloalkyl-C(O)O--, substituted
cycloalkyl-C(O)O--, cycloalkenyl-C(O)O--, substituted
cycloalkenyl-C(O)O--, heteroaryl-C(O)O--, substituted
heteroaryl-C(O)O--, heterocyclic-C(O)O--, and substituted
heterocyclic-C(O)O--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
Amino" refers to the group --NH.sub.2.
"Substituted amino" refers to the group --NR'R'' where R' and R''
are independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, substituted
heterocyclic, --SO.sub.2-alkyl, --SO.sub.2-substituted alkyl,
--SO.sub.2-alkenyl, --SO.sub.2-substituted alkenyl,
--SO.sub.2-cycloalkyl, --SO.sub.2-substituted cycloalkyl,
--SO.sub.2-cycloalkenyl, --SO.sub.2-substituted cylcoalkenyl,
--SO.sub.2-aryl, --SO.sub.2-substituted aryl,
--SO.sub.2-heteroaryl, --SO.sub.2-substituted heteroaryl,
--SO.sub.2-heterocyclic, and --SO.sub.2-substituted heterocyclic
and wherein R' and R'' are optionally joined, together with the
nitrogen bound thereto to form a heterocyclic or substituted
heterocyclic group, provided that R' and R'' are both not hydrogen,
and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein. When R' is hydrogen and R'' is
alkyl, the substituted amino group is sometimes referred to herein
as alkylamino. When R' and R'' are alkyl, the substituted amino
group is sometimes referred to herein as dialkylamino. When
referring to a monosubstituted amino, it is meant that either R' or
R'' is hydrogen but not both. When referring to a disubstituted
amino, it is meant that neither R' nor R'' are hydrogen.
"Aminocarbonyl" refers to the group --C(O)NR'R'' where R' and R''
are independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Aminothiocarbonyl" refers to the group --C(S)NR'R'' where R' and
R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Aminocarbonylamino" refers to the group --NRC(O)NR' R'' where R is
hydrogen or alkyl and R' and R'' are independently selected from
the group consisting of hydrogen, alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic, and where R' and R''
are optionally joined together with the nitrogen bound thereto to
form a heterocyclic or substituted heterocyclic group, and wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
"Aminothiocarbonylamino" refers to the group --NRC(S)NR'R'' where R
is hydrogen or alkyl and R' and R'' are independently selected from
the group consisting of hydrogen, alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic, and where R' and R''
are optionally joined together with the nitrogen bound thereto to
form a heterocyclic or substituted heterocyclic group, and wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
"Aminocarbonyloxy" refers to the group --O--C(O)NR'R'' where R' and
R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Aminosulfonyl" refers to the group --SO.sub.2NR'R'' where R' and
R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Aminosulfonyloxy" refers to the group --O--SO.sub.2NR'R'' where R'
and R'' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Aminosulfonylamino" refers to the group --NR--SO.sub.2NR'R'' where
R is hydrogen or alkyl and R' and R'' are independently selected
from the group consisting of hydrogen, alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,
substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted cycloalkyenyl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic, and where R' and R''
are optionally joined together with the nitrogen bound thereto to
form a heterocyclic or substituted heterocyclic group, and wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkyenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic and substituted
heterocyclic are as defined herein.
"Amidino" refers to the group --C(.dbd.NR''')R'R'' where R', R'',
and R''' are independently selected from the group consisting of
hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic, and where R' and R'' are optionally joined together
with the nitrogen bound thereto to form a heterocyclic or
substituted heterocyclic group, and wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Aniline" refers to C.sub.6H.sub.5NH.sub.2, and consists of a
phenyl ring attached to an amino group. As used herein, the amino
group is para to a fluorophore, as is illustrated as follows:
##STR00007##
wherein X is a fluorophore, preferably a xanthene derivative, most
preferably a rhodamine or rhodol.
"Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of
from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or
multiple condensed rings (e.g., naphthyl or anthryl) which
condensed rings may or may not be aromatic (e.g.,
2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like)
provided that the point of attachment is at an aromatic carbon
atom. Preferred aryl groups include phenyl and naphthyl.
"Substituted aryl" refers to aryl groups which are substituted with
1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents
selected from the group consisting of alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,
substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted
amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,
aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,
aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted
aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio,
carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl
ester)oxy, cyano, cycloalkyl, substituted cycloalkyl,
cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,
substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyl,
cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio,
substituted cycloalkenylthio, guanidino, substituted guanidino,
halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy,
substituted heteroaryloxy, heteroarylthio, substituted
heteroarylthio, heterocyclic, substituted heterocyclic,
heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,
substituted heterocyclylthio, nitro, SO.sub.3H, substituted
sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, and substituted
alkylthio, wherein said substituents are defined herein.
"Aryloxy" refers to the group --O-aryl, where aryl is as defined
herein, that includes, by way of example, phenoxy and
naphthoxy.
"Substituted aryloxy" refers to the group --O-(substituted aryl),
where substituted aryl is as defined herein.
"Arylthio" refers to the group --S-aryl, where aryl is as defined
herein.
"Substituted arylthio" refers to the group --S-(substituted aryl),
where substituted aryl is as defined herein.
"Alkenyl" refers to alkenyl groups having from 2 to 6 carbon atoms
and preferably 2 to 4 carbon atoms and having at least 1 and
preferably from 1 to 2 sites of alkenyl unsaturation. Such groups
are exemplified, for example, by vinyl, allyl, but-3-en-1-yl, and
propenyl.
"Substituted alkenyl" refers to alkenyl groups having from 1 to 3
substituents, and preferably 1 to 2 substituents, selected from the
group consisting of alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein and with the proviso
that any hydroxy substitution is not attached to a vinyl
(unsaturated) carbon atom.
"Alkynyl" refers to alkynyl groups having from 2 to 6 carbon atoms
and preferably 2 to 3 carbon atoms and having at least 1 and
preferably from 1 to 2 sites of alkynyl unsaturation.
"Substituted alkynyl" refers to alkynyl groups having from 1 to 3
substituents, and preferably 1 to 2 substituents, selected from the
group consisting of alkoxy, substituted alkoxy, acyl, acylamino,
acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein and with the proviso
that any hydroxy substitution is not attached to an acetylenic
carbon atom.
"Carbonyl" refers to the divalent group --C(O)-- which is
equivalent to --C(.dbd.O)--.
"Carboxyl" or "carboxy" refers to --COOH or salts thereof.
"Carboxyl alkyl" or "carboxyalkyl" refers to the groups
--(CH.sub.2).sub.nCOOH, wherein n is an integer from 1 to 6.
"Carboxyl ester" or "carboxy ester" refers to the groups
--C(O)O-alkyl, --C(O)O-substituted alkyl, --C(O)O-alkenyl,
--C(O)O-substituted alkenyl, --C(O)O-alkynyl, --C(O)O-substituted
alkynyl, --C(O)O-aryl, --C(O)O-substituted aryl,
--C(O)O-cycloalkyl, --C(O)O-substituted cycloalkyl,
--C(O)O-cycloalkenyl, --C(O)O-substituted cycloalkenyl,
--C(O)O-heteroaryl, --C(O)O-substituted heteroaryl,
--C(O)O-heterocyclic, and --C(O)O-substituted heterocyclic, wherein
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,
substituted alkynyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, heterocyclic, and substituted
heterocyclic are as defined herein.
"(Carboxyl ester)amino" refers to the group --NR--C(O)O-alkyl,
substituted --NR--C(O)O-alkyl, --NR--C(O)O-alkenyl,
--NR--C(O)O-substituted alkenyl, --NR--C(O)O-alkynyl,
--NR--C(O)O-substituted alkynyl, --NR--C(O)O-aryl,
--NR--C(O)O-substituted aryl, --NR--C(O)O-cycloalkyl,
--NR--C(O)O-substituted cycloalkyl, --NR--C(O)O-cycloalkenyl,
--NR--C(O)O-substituted cycloalkenyl, --NR--C(O)O-heteroaryl,
--NR--C(O)O-substituted heteroaryl, --NR--C(O)O-heterocyclic, and
--NR--C(O)O-substituted heterocyclic, wherein R is alkyl or
hydrogen, and wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic, and substituted heterocyclic are as defined
herein.
"(Carboxyl ester)oxy" refers to the group --O--C(O)O-alkyl,
substituted --O--C(O)O-alkyl, --O--C(O)O-alkenyl,
--O--C(O)O-substituted alkenyl, --O--C(O)O-alkynyl,
--O--C(O)O-substituted alkynyl, --O--C(O)O-aryl,
--O--C(O)O-substituted aryl, --O--C(O)O-cycloalkyl,
--O--C(O)O-substituted cycloalkyl, --O--C(O)O-cycloalkenyl,
--O--C(O)O-substituted cycloalkenyl, --O--C(O)O-heteroaryl,
--O--C(O)O-substituted heteroaryl, --O--C(O)O-heterocyclic, and
--O--C(O)O-substituted heterocyclic, wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic, and substituted heterocyclic are as
defined herein.
"Cyano" refers to the group CN.
"Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon
atoms having single or multiple cyclic rings including fused,
bridged, and spiro ring systems. Examples of suitable cycloalkyl
groups include, for instance, adamantyl, cyclopropyl, cyclobutyl,
cyclopentyl, and cyclooctyl.
"Cycloalkenyl" refers to non-aromatic cyclic alkyl groups of from 3
to 10 carbon atoms having single or multiple cyclic rings and
having at least one >C.dbd.C< ring unsaturation and
preferably from 1 to 2 sites of >C.dbd.C< ring
unsaturation.
"Substituted cycloalkyl" and "substituted cycloalkenyl" refers to a
cycloalkyl or cycloalkenyl group having from 1 to 5 or preferably 1
to 3 substituents selected from the group consisting of oxo,
thione, alkyl, substituted alkyl, alkenyl, substituted alkenyl,
alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl,
acylamino, acyloxy, amino, substituted amino, aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy,
aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy,
substituted aryloxy, arylthio, substituted arylthio, carboxyl,
carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,
cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted
cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy,
substituted cycloalkenyloxy, cycloalkenylthio, substituted
cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy,
heteroaryl, substituted heteroaryl, heteroaryloxy, substituted
heteroaryloxy, heteroarylthio, substituted heteroarylthio,
heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted heterocyclyloxy, heterocyclylthio, substituted
heterocyclylthio, nitro, SO.sub.3H, substituted sulfonyl,
sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,
wherein said substituents are defined herein.
"Cycloalkyloxy" refers to --O-cycloalkyl.
"Substituted cycloalkyloxy" refers to --O-(substituted
cycloalkyl).
"Cycloalkylthio" refers to --S-cycloalkyl.
"Substituted cycloalkylthio" refers to --S-(substituted
cycloalkyl).
"Cycloalkenyloxy" refers to --O-cycloalkenyl.
"Substituted cycloalkenyloxy" refers to --O-(substituted
cycloalkenyl).
"Cycloalkenylthio" refers to --S-cycloalkenyl.
"Substituted cycloalkenylthio" refers to --S-(substituted
cycloalkenyl).
"Guanidino" refers to the group --NHC(.dbd.NH)NH.sub.2.
"Substituted guanidino" refers to
--NR.sup.13C(.dbd.NR.sup.13)N(R.sup.13).sub.2 where each R.sup.13
is independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, heterocyclic, and substituted heterocyclic
and two R.sup.13 groups attached to a common guanidino nitrogen
atom are optionally joined together with the nitrogen bound thereto
to form a heterocyclic or substituted heterocyclic group, provided
that at least one R.sup.13 is not hydrogen, and wherein said
substituents are as defined herein.
"H" indicates hydrogen.
"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.
"Hydroxy" or "hydroxyl" refers to the group --OH.
"Heteroaryl" refers to an aromatic group of from 1 to 10 carbon
atoms and 1 to 4 heteroatoms selected from the group consisting of
oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups
can have a single ring (e.g., pyridinyl or furyl) or multiple
condensed rings (e.g., indolizinyl or benzothienyl) wherein the
condensed rings may or may not be aromatic and/or contain a
heteroatom provided that the point of attachment is through an atom
of the aromatic heteroaryl group. In one embodiment, the nitrogen
and/or the sulfur ring atom(s) of the heteroaryl group are
optionally oxidized to provide for the N-oxide (N.fwdarw.O),
sulfinyl, or sulfonyl moieties. Preferred heteroaryls include
pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
"Substituted heteroaryl" refers to heteroaryl groups that are
substituted with from 1 to 5, preferably 1 to 3, or more preferably
1 to 2 substituents selected from the group consisting of the same
group of substituents defined for substituted aryl.
"Heteroaryloxy" refers to --O-heteroaryl.
"Substituted heteroaryloxy" refers to the group --O-(substituted
heteroaryl).
"Heteroarylthio" refers to the group --S-heteroaryl.
"Substituted heteroarylthio" refers to the group --S-(substituted
heteroaryl).
"Heterocycle" or "heterocyclic" or "heterocycloalkyl" or
"heterocyclyl" refers to a saturated or unsaturated group having a
single ring or multiple condensed rings, including fused bridged
and spiro ring systems, from 1 to 10 carbon atoms and from 1 to 4
hetero atoms selected from the group consisting of nitrogen, sulfur
or oxygen within the ring wherein, in fused ring systems, one or
more the rings can be cycloalkyl, aryl or heteroaryl provided that
the point of attachment is through the non-aromatic ring. In one
embodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic
group are optionally oxidized to provide for the N-oxide, sulfinyl,
and sulfonyl moieties.
"Substituted heterocyclic" or "substituted heterocycloalkyl" or
"substituted heterocyclyl" refers to heterocyclyl groups that are
substituted with from 1 to 5, or preferably 1 to 3 of the same
substituents as defined for substituted cycloalkyl.
"Heterocyclyloxy" refers to the group --O-heterocyclyl.
"Substituted heterocyclyloxy" refers to the group --O-(substituted
heterocyclyl).
"Heterocyclylthio" refers to the group --S-- heterocyclyl.
"Substituted heterocyclylthio" refers to the group --S-(substituted
heterocyclyl).
Examples of heterocycle and heteroaryls include, but are not
limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine,
pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,
dihydroindole, indazole, purine, quinolizine, isoquinoline,
quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline,
cinnoline, pteridine, carbazole, carboline, phenanthridine,
acridine, phenanthroline, isothiazole, phenazine, isoxazole,
phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine,
piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,
4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine,
thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also
referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl,
piperidinyl, pyrrolidine, and tetrahydrofuranyl.
"Hydrazinyl" refers to the group --NHNH.sub.2-- or .dbd.NNH--.
"Substituted hydrazinyl" refers to a hydrazinyl group, wherein a
non-hydrogen atom, such as an alkyl group, is appended to one or
both of the hydrazinyl amine groups. An example of substituted
hydrazinyl is --N(alkyl)-NH.sub.2 or
.dbd.N.sup.+(alkyl)-NH.sub.2.
"Nitro" refers to the group --NO.sub.2.
"Oxo" refers to the atom (.dbd.O) or (--O.sup.-).
"Spirocyclyl" refers to divalent saturated cyclic group from 3 to
10 carbon atoms having a cycloalkyl or heterocyclyl ring with a
spiro union (the union formed by a single atom which is the only
common member of the rings) as exemplified by the following
structure:
##STR00008##
"Sulfonyl" refers to the divalent group --S(O).sub.2--.
"Substituted sulfonyl" refers to the group --SO.sub.2-alkyl,
--SO.sub.2-substituted alkyl, --SO.sub.2-alkenyl,
--SO.sub.2-substituted alkenyl, --SO.sub.2-cycloalkyl,
--SO.sub.2-substituted cycloalkyl, --SO.sub.2-cycloalkenyl,
--SO.sub.2-substituted cylcoalkenyl, --SO.sub.2-aryl,
--SO.sub.2-substituted aryl, --SO.sub.2-heteroaryl,
--SO.sub.2-substituted heteroaryl, --SO.sub.2-heterocyclic,
--SO.sub.2-substituted heterocyclic, wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein. Substituted sulfonyl includes groups such as
methyl-SO.sub.2--, phenyl-SO.sub.2--, and
4-methylphenyl-SO.sub.2--.
"Sulfonyloxy" refers to the group --OSO.sub.2-alkyl,
--OSO.sub.2-substituted alkyl, --OSO.sub.2-alkenyl,
--OSO.sub.2-substituted alkenyl, --OSO.sub.2-cycloalkyl,
--OSO.sub.2-substituted cycloalkyl, --OSO.sub.2-cycloalkenyl,
--OSO.sub.2-substituted cylcoalkenyl, --OSO.sub.2-aryl,
--OSO.sub.2-substituted aryl, --OSO.sub.2-heteroaryl,
--OSO.sub.2-substituted heteroaryl, --OSO.sub.2-heterocyclic,
--OSO.sub.2-substituted heterocyclic, wherein alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, heterocyclic and substituted heterocyclic are as
defined herein.
"Thioacyl" refers to the groups H--C(S)--, alkyl-C(S)--,
substituted alkyl-C(S)--, alkenyl-C(S)--, substituted
alkenyl-C(S)--, alkynyl-C(S)--, substituted alkynyl-C(S)--,
cycloalkyl-C(S)--, substituted cycloalkyl-C(S)--,
cycloalkenyl-C(S)--, substituted cycloalkenyl-C(S)--, aryl-C(S)--,
substituted aryl-C(S)--, heteroaryl-C(S)--, substituted
heteroaryl-C(S)--, heterocyclic-C(S)--, and substituted
heterocyclic-C(S)--, wherein alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
heterocyclic and substituted heterocyclic are as defined
herein.
"Thiol" refers to the group --SH.
"Thiocarbonyl" refers to the divalent group --C(S)-- which is
equivalent to --C(.dbd.S)--.
"Thione" refers to the atom (.dbd.S).
"Alkylthio" refers to the group --S-alkyl wherein alkyl is as
defined herein.
"Substituted alkylthio" refers to the group --S-(substituted
alkyl), wherein substituted alkyl is as defined herein.
A dashed line projecting from a substituent, such as:
##STR00009## indicates the point of attachment to the base
molecule. For a fused ring, dashed lines indicate portions of the
base molecule where the fused ring is attached, such as:
##STR00010## wherein the full molecule could have the
structure:
##STR00011##
Unless indicated otherwise, the nomenclature of substituents that
are not explicitly defined herein are arrived at by naming the
terminal portion of the functionality followed by the adjacent
functionality toward the point of attachment. For example, the
substituent "arylalkyloxycarbonyl" refers to the group
(aryl)-(alkyl)-O--C(O)--.
It is understood that in all substituted groups defined above,
polymers arrived at by defining substituents with further
substituents to themselves (e.g., substituted aryl having a
substituted aryl group as a substituent which is itself substituted
with a substituted aryl group, which is further substituted by a
substituted aryl group etc.) are not intended for inclusion herein.
In such cases, the maximum number of such substitutions is three.
For example, serial substitutions of substituted aryl groups with
two other substituted aryl groups are limited to -substituted
aryl-(substituted aryl)-substituted aryl.
Similarly, it is understood that the above definitions are not
intended to include impermissible substitution patterns (e.g.,
methyl substituted with 5 fluoro groups). Such impermissible
substitution patterns are well known to the skilled artisan.
The pH-sensitive fluorescent dye compounds disclosed herein may
exist in unsolvated forms as well as solvated forms, including
hydrated forms. These compounds may exist in multiple crystalline
or amorphous forms. In general, all physical forms are equivalent
for the uses described herein and are intended to be within the
scope of the present disclosure. The dye compounds disclosed herein
may possess asymmetric carbon atoms (i.e., chiral centers) or
double bonds; the racemates, diastereomers, geometric isomers and
individual isomers of the compounds described herein are within the
scope of the present disclosure. The dye compounds described herein
may be prepared as a single isomer or as a mixture of isomers.
Where substituent groups are specified by their conventional
chemical formulae and are written from left to right, they equally
encompass the chemically identical substituents, which would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is intended to also recite --OCH.sub.2--.
It will be understood that the chemical structures that are used to
define the dye compounds disclosed herein are each representations
of one of the possible resonance structures by which each given
structure can be represented. Further, it will be understood that
by definition, resonance structures are merely a graphical
representation used by those of skill in the art to represent
electron delocalization, and that the present disclosure is not
limited in any way by showing one particular resonance structure
for any given structure.
Where a disclosed compound includes a conjugated ring system,
resonance stabilization may permit a formal electronic charge to be
distributed over the entire molecule. While a particular charge may
be depicted as localized on a particular ring system, or a
particular heteroatom, it is commonly understood that a comparable
resonance structure can be drawn in which the charge may be
formally localized on an alternative portion of the compound.
The term "carrier molecule" as used herein, refers to a biological
or a non-biological component that is or becomes covalently bonded
to a pH-sensitive fluorescent dye compound disclosed herein. Such
components include, but are not limited to, an amino acid, a
peptide, a protein, a polysaccharide, a nucleoside, a nucleotide,
an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a
hormone, a lipid, a lipid assembly, a synthetic polymer, a
polymeric microparticle, a biological cell, a virus and
combinations thereof. Included is one embodiment in which carrier
molecules comprise an organic moiety having at least 4 plural
valent atoms and often more than 10 plural valent atoms (i.e.,
atoms other than hydrogen and halo), e.g. at least 15 such atoms,
as in the case of moieties having at least 20 such atoms.
The term "conjugated substance" or "S.sub.c" refers to a carrier
molecule or solid support.
The term "detectable response" as used herein refers to an
occurrence of or a change in, a signal that is directly or
indirectly detectable either by observation or by instrumentation.
Typically, the detectable response is an optical response resulting
in a change in the wavelength distribution patterns or intensity of
absorbance or fluorescence or a change in light scatter,
fluorescence lifetime, fluorescence polarization, or a combination
of the above parameters.
The term "dye" as used herein refers to a compound that emits light
to produce an observable detectable signal.
The term "electron donating group" or "EDG" refers to a substituent
with lone electron pairs that is adjacent to an aromatic ring, such
as phenyl, and increases electron density on the ring through a
resonance donating effect. Electron donating groups of the present
disclosure include, for example, alkoxy, substituted alkoxy, amino,
substituted amino, halogen, alkylthio, acylamino, and (carboxyl
ester)oxy. Alkoxy is a particular EDG. Substituted alkoxy is
another particular EDG. Also to be mentioned is dialkylamino. A
further example is dialkylamino having a substituted alkyl group.
Preferred EDGs are --OCH.sub.3, --NH.sub.2, --NHCH.sub.3,
--N(CH.sub.3).sub.2, and --N(CH.sub.2CH.sub.3).sub.2, particularly
--OCH.sub.3, --NH.sub.2, --NHCH.sub.3, --N(CH.sub.3).sub.2 and
--N(CH.sub.2CH.sub.3).sub.2. Also to be mentioned are alkoxy,
alkythio and dialkylamino, in any of those instances having an
alkyl substituent in which the alkyl part is substituted by a
moiety or -L.sub.R-S.sub.c. The specification also discloses
specific compounds or compound classes which include other EDGs
than those with a lone pair of electrons adjacent an aromatic
ring.
"Fluorescent pH-sensitive dye," "pH-sensitive fluorescent dye," and
"fluorescent pH sensor dye" are equivalent and are used
interchangeably to refer to a compound whose fluorescent spectrum
or intensity is affected by pH.
The term "fluorophore" or "fluorogenic" as used herein refers to a
composition that is inherently fluorescent or demonstrates a change
in fluorescence upon protonation, or binding to a biological
compound or metal ion, or metabolism by an enzyme. Preferred
fluorophores of the present disclosure include fluorescent dyes
having a high quantum yield in aqueous media. Exemplary
fluorophores include xanthene derivatives, preferably rhodamines
and rhodols. The fluorophores disclosed herein may be substituted
to alter the solubility, spectral properties or physical properties
of the fluorophore.
The term "linker" or "L", as used herein, refers to a single
covalent bond or a moiety comprising series of stable covalent
bonds, the moiety often incorporating 1-40 plural valent atoms
selected from the group consisting of C, N, O, S and P that
covalently attach the fluorogenic or fluorescent compounds to
another moiety such as a chemically reactive group or a biological
and non-biological component. The number of plural valent atoms in
a linker may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
25, 30 or a larger number up to 40 or more. A linker may be linear
or non-linear; some linkers have pendant side chains or pendant
functional groups, or both. Examples of such pendant moieties are
hydrophilicity modifiers, for example solubilizing groups like,
e.g. sulfo (--SO.sub.3H or --SO.sub.3.sup.-). In certain
embodiments, L is composed of any combination of single, double,
triple or aromatic carboncarbon bonds, carbonnitrogen bonds,
nitrogennitrogen bonds, carbonoxygen bonds and carbonsulfur bonds.
Exemplary linking members include a moiety that includes C(O)NH--,
--C(O)O--, --NH--, --S--, --O--, and the like. Linkers may, by way
of example, consist of a combination of moieties selected from
alkyl; C(O)NH--; --C(O)O--; --NH--; --S--; --O--; --C(O)--;
--S(O)-- where n is 0, 1 or 2; --O--; 5- or 6-membered monocyclic
rings; and optional pendant functional groups, for example sulfo,
hydroxy and carboxy. The moiety formed by a linker bonded to a
reactive group (R.sub.x) may be designated -L-R.sub.x. The reactive
group may be reacted with a substance reactive therewith, whereby
the linker becomes bonded to a conjugated substance (S.sub.c); in
this case, the linker typically contains a residue of a reactive
group (e.g. the carbonyl group of an ester) and may be designated
"-L.sub.R". A "cleavable linker" is a linker that has one or more
cleavable groups that may be broken by the result of a reaction or
condition. The term "cleavable group" refers to a moiety that
allows for release of a portion, e.g., a fluorogenic or fluorescent
moiety, of a conjugate from the remainder of the conjugate by
cleaving a bond linking the released moiety to the remainder of the
conjugate. Such cleavage is either chemical in nature, or
enzymatically mediated. Exemplary enzymatically cleavable groups
include natural amino acids or peptide sequences that end with a
natural amino acid.
In addition to enzymatically cleavable groups, it is within the
scope of the present invention to include one or more sites that
are cleaved by the action of an agent other than an enzyme.
Exemplary non-enzymatic cleavage agents include, but are not
limited to, acids, bases, light (e.g., nitrobenzyl derivatives,
phenacyl groups, benzoin esters), and heat. Many cleavable groups
are known in the art. See, for example, Jung et al., Biochem.
Biophys. Acta, 761:152-162 (1983); Joshi et al., J. Biol. Chem.,
265:14518-14525 (1990); Zarling et al., J. Immunol., 124:913-920
(1980); Bouizar et al., Eur. J. Biochem., 155:141-147 (1986); Park
et al., J. Biol. Chem., 261:205-210 (1986); Browning et al., J.
Immunol., 143:1859-1867 (1989). Moreover a broad range of
cleavable, bifunctional (both homo- and hetero-bifunctional) spacer
arms are commercially available.
An exemplary cleavable group, such as an ester, is a cleavable
group that may be cleaved by a reagent, e.g., sodium hydroxide,
resulting in a carboxylate-containing fragment and a
hydroxyl-containing product.
The linker may be used to attach the pH-sensitive fluorescent dye
compound to another component of a conjugate, such as a targeting
moiety (e.g., antibody, ligand, non-covalent protein-binding group,
etc.), an analyte, a biomolecule, a drug and the like.
In certain embodiments, -L- is of the formula
-L1-(L2).sub.p-(L3).sub.r-, wherein p is 0 or 1; r is 0 or 1; L1 is
a bond, --CONH--, --COO--, or a moiety comprising at least two
amino acids; L2 is --(CH.sub.2)--,
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.s--CH.sub.2CH.sub.2--,
or alkylene having from 1 to 30 carbon atoms and unsubstituted or
substituted by at least one R.sup.a, e.g. 1, 2, 3, 4, 5 or 6
R.sup.a; L3 is --CONH--(CH.sub.2).sub.t--,
--COO--(CH.sub.2).sub.t-- or a moiety comprising at least two amino
acids, wherein: r is from 1 to 30, e.g., 1 to 20 as in the case of
1 to 10, such as 1, 2, 3, 4, 5 or 6; s is 0, 1, 2, 3, 4, 5, 6, 7,
8, 9 or 10, e.g., 1 to 7; t is from 1 to 30, e.g. 1 to 20 as in the
case of 1 to 10, such as 1, 2, 3, 4, 5 or 6; R.sup.a is sulfo
(--SO.sub.3H and/or --SO.sub.3.sup.-), hydroxy, carboxy or amino,
particularly sulfo.
In certain embodiments, the total number of carbon atoms comprised
in alkylene moieties in L is no more than 40, e.g., up to 35, 30,
25, 20, 15 or 10. In certain embodiments, there is only a single
one of L1 and L3 which comprises a moiety comprising at least two
amino acids.
In certain embodiments, p and r are both 0. In certain embodiments,
p is 1 and r is 0. In another certain embodiments, p and r are both
1.
In certain embodiments, L1 is a bond, --CONH-- or --COO--. In
certain compounds L1 is a bond. In certain others, L1 is
--CONH--.
In certain embodiments, L2 is --(CH.sub.2).sub.u--, where u is from
1 to 10, e.g., 1, 2, 3, 4, 5 or 6. In certain embodiments, L2 is
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.s--CH.sub.2CH.sub.2--
where s is from 1 to 7. In certain embodiments, L2 is alkylene
having from 1 to 10 carbon atoms, e.g., 1, 2, 3, 4, 5 or 6 carbon
atoms, and which is unsubstituted or substituted by 1, 2, 3, 4, 5
or 6 sulfo groups, e.g., 1 to 4 sulfo groups. For all L2 moieties
mentioned in this paragraph, L1 is --CONH-- in a particular class
of compounds. For all L2 moieties mentioned in this paragraph and
all -L1-L2- combinations mentioned in this paragraph, r is 0 in one
class of compounds.
In certain embodiments, (r+t) is from 1 to 30, e.g., 1 to 20 as in
the case of 1 to 10, such as 1, 2, 3, 4, 5 or 6, for example.
Exemplary linkers include, but are not limited to, the following: a
single covalent bond (for example between alkyl and a carboxy group
or ester of a carboxy group, or other reactive group);
aminocarbonyl (for example linking an alkyl group to a conjugated
lipophilic moiety); a PEG-NH--CO-moiety (for example linking an
alkyl group to an NHS-ester or other reactive group); an
alkylaminocarbonyl group (for example linking an alkyl group to an
NHS-ester, amine or other reactive group); an alkylaminocarbonyl
group having a pendant group comprising sulfo--e.g. a pendant
sulfoalkyl group (for example linking an alkyl group to NHS-ester
or other reactive group or to a lipophilic group); or a single
covalent bond linking an alkyl group to a reactive group such as a
carboxy group or ester thereof.
The terms "patient," "subject" or "individual" refer to mammals and
includes humans and non-human mammals, such as monkeys, dogs, cats,
pocket pets, horses, cows, pigs or rats.
The terms "protein" and "polypeptide" are used herein in a generic
sense to include polymers of amino acid residues of any length. The
term "peptide" is used herein to refer to polypeptides having less
than 250 amino acid residues, typically less than 100 amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residues are an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
The term "reactive group" (or "R.sub.x"), as used herein, refers to
a group that is capable of reacting with another chemical group to
form a covalent bond, i.e., is covalently reactive under suitable
reaction conditions, and generally represents a point of attachment
for another substance. The reactive group is a moiety, such as
carboxylic acid or succinimidyl ester, on the compounds of the
present disclosure that is capable of chemically reacting with a
functional group on a different compound to form a covalent
linkage. Reactive groups generally include nucleophiles,
electrophiles and photoactivatable groups.
Exemplary reactive groups include, but are not limited to, olefins,
acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,
ketones, carboxylic acids, esters, amides, cyanates, isocyanates,
thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,
hydrazides, diazo, diazonium, nitro, nitriles, mercaptans,
sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,
sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic
acids isonitriles, amidines, imides, imidates, nitrones,
hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids,
allenes, ortho esters, sulfites, enamines, ynamines, ureas,
pseudoureas, semicarbazides, carbodiimides, carbamates, imines,
azides, azo compounds, azoxy compounds, and nitroso compounds.
Reactive functional groups also include those used to prepare
bioconjugates, e.g., N-hydroxysuccinimide esters, maleimides,
succinimidyl esters (SE), sulfodichlorophenol (SDP) esters,
sulfotetrafluorophenol (STP) esters, tetrafluorophenol (TFP)
esters, acetoxymethoxy (AM) esters, nitrilotriacetic acids (NTA),
aminodextrans, DIBO-amines and the like. Methods to prepare each of
these functional groups are well known in the art and their
application to or modification for a particular purpose is within
the ability of one of skill in the art (see, for example, Sandler
and Karo, eds., Organic Functional Group Preparations, Academic
Press, San Diego, 1989).
The term "salt" refers to acceptable salts of a compound, which
salts are derived from a variety of organic and inorganic counter
ions well known in the art and include, by way of example only,
sodium, potassium, calcium, magnesium, ammonium, and
tetraalkylammonium; and when the molecule contains a basic
functionality, salts of organic or inorganic acids, such as
hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,
and oxalate.
The term "sample," as used herein, refers to any material that may
contain an analyte of interest or cells. Typically, the sample is a
live cell or a biological fluid that comprises endogenous host
cells. Alternatively, the sample may be a buffer solution or an
environmental sample for which pH determination is needed. The
sample may be in an aqueous solution, a viable cell culture or
immobilized on a solid or semi-solid surface such as a
polyacrylamide gel, membrane blot or on a microarray.
The term "solid support," as used herein, refers to a matrix or
medium that is substantially insoluble in liquid phases and capable
of binding a molecule or particle of interest. Solid supports
suitable for use herein include semi-solid supports and are not
limited to a specific type of support. Useful solid supports
include solid and semi-solid matrixes, such as aerogels and
hydrogels, resins, beads, biochips (including thin film coated
biochips), microfluidic chip, a silicon chip, multi-well plates
(also referred to as microtitre plates or microplates), membranes,
conducting and nonconducting metals, glass (including microscope
slides) and magnetic supports. More specific examples of useful
solid supports include silica gels, polymeric membranes, particles,
derivatized plastic films, glass beads, cotton, plastic beads,
alumina gels, polysaccharides such as Sepharose.RTM.,
poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose,
agar, cellulose, dextran, starch, FICOLL.RTM., heparin, glycogen,
amylopectin, mannan, inulin, nitrocellulose, diazocellulose,
polyvinylchloride, polypropylene, polyethylene (including
poly(ethylene glycol)), nylon, latex bead, magnetic bead,
paramagnetic bead, superparamagnetic bead, starch and the like.
The terms "stereoisomer" or "stereoisomers" refer to compounds that
differ in the chirality of one or more stereocenters. Stereoisomers
include enantiomers and diastereomers.
The term "tautomer" refers to alternate forms of a compound that
differ in the position of a proton, such as enol-keto and
imine-enamine tautomers, or the tautomeric forms of heteroaryl
groups containing a ring atom attached to both a ring --NH-- moiety
and a ring .dbd.N-- moiety such as pyrazoles, imidazoles,
benzimidazoles, triazoles, and tetrazoles.
The terms "treating" or "treatment" of a disease in a patient refer
to 1) preventing the disease from occurring in a patient that is
predisposed or does not yet display symptoms of the disease; 2)
inhibiting the disease or arresting its development; or 3)
ameliorating or causing regression of the disease.
Dye Compounds and Compositions:
In general, for ease of understanding the present disclosure, the
pH-sensitive fluorescent dye compounds and corresponding
substituents will first be described in detail, followed by various
methods in which the pH-sensitive fluorescent dye compounds of the
present disclosure are useful, which is followed by exemplary
methods of use and synthesis of certain novel dye compounds that
are particularly advantageous for use with the methods provided
herein.
The pH-sensitive fluorescent dye compounds disclosed herein are
useful for monitoring or detecting pH in a sample. For example, we
have found that by introducing an electron donating group (EDG)
into the 4-amino-2-hydroxyphenyl ring of a pH-sensitive fluorescent
dye compound that we were able to tune the fluorescent properties
of the pH-sensitive fluorescent dye compound (See, structural
formulae I and II). In particular we were able to tune the pKa
value and obtain a pH-sensitive dye compound with a pKa value
compatible with live cell intracellular applications. We also found
that replacing the hydroxyl at position R.sup.1 with an alkoxy or
thioalkyl moiety not only increased the stability of the dye
compound in an aqueous environment but also resulted in a compound
that was pH sensitive, an unexpected advantage in view of the
teaching by Smith et al. (supra). Advantageously, the addition of a
dialkylamino group to the aniline moiety at position R.sup.3
resulted in the unexpected advantage of yielding pKa values in the
physiological range. In certain embodiments, the dialkylamino group
is diethylamino. In certain embodiments, the dialkylamino group is
dimethylamino.
In certain embodiments, the pKa value is about 6 to about 7.
Without wishing to be bound by a theory, the pKa of the amino group
of the aniline moiety of the compounds of structural formulae I and
II appears to depend on the ability of the aromatic system to share
a lone electron pair on the oxygen atom. This ability is affected
by additional functional groups introduced into the aromatic system
and thus, the pKa is tuned by adding EDG groups to pH sensitive
dyes comprising an electron rich aniline moiety.
In certain embodiments, the sample to be analyzed includes live
cells or a biological fluid, including cytosol that comprises
endogenous host cell proteins, buffer solutions and environmental
samples. Therefore, the pH-sensitive fluorescent dye compounds
disclosed herein are useful for monitoring or determining pH
changes and those events directly and indirectly associated with a
change in pH. Monitoring of the pH may also be accomplished in live
cells wherein the present pH-sensitive fluorescent dye compounds
are internalized by live cells through a number of different
mechanisms, including both passive and cell mediated mechanisms.
For example, the present pH-sensitive fluorescent dye compounds may
comprise a lipophilic group such as an acetoxymethoxy (AM) or
acetate ester that allows for entry across the live cell membrane.
Once inside the cells, nonspecific esterases cleave the AM or
acetate ester resulting in a charged molecule that is well retained
in the cell. Alternatively, the present pH-sensitive fluorescent
dye compounds may be conjugated to a carrier molecule that allows
the dye compound to be taken up by live cells. Examples include
internalization during phagocytosis, wherein the pH-sensitive
fluorescent dye compounds are conjugated to bacterial particles or
other proteins (or peptides) that induce phagocytosis by
macrophages or monocytes; or up-take through receptor
internalization when the present pH-sensitive fluorescent dye
compounds are conjugated to a carrier molecule that binds a
receptor and thus induces internalization.
The pH-sensitive fluorescent dye compounds disclosed herein
function as reporter molecules to confer a detectable signal,
directly or indirectly, to the sample as a result of a change in
pH. This results in the ability to measure and monitor pH changes
in a sample to directly and indirectly detect specific events
associated with a change in pH.
Where the detectable response is a fluorescence response, it is
typically a change in fluorescence, such as a change in the
intensity, excitation or emission wavelength, distribution of
fluorescence, fluorescence lifetime, fluorescence polarization, or
a combination thereof. In certain embodiments, the detectable
optical response upon protonation is a change in fluorescence
intensity that is greater than approximately 150% relative to the
same dye compound wherein the aniline moiety is not protonated on
the nitrogen. Preferably, the change in fluorescence intensity is
greater than 5-fold, and more preferably more than 10-fold.
The pH-sensitive fluorescent dye compounds provided herein may
comprise a fluorophore that may be any rhodamine or rhodol
fluorophore known to one skilled in the art that fluoresces in the
red portion of the UV/VIS spectrum. Preferably, the fluorophore is
quenched, or substantially non-fluorescent, until the nitrogen on
the aniline moiety is protonated.
In certain embodiments, the pH-sensitive fluorescent dye compounds
are independently substituted by substituents selected from the
group consisting of hydrogen, halogen, amino, substituted amino,
alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, alkoxy, sulfo, reactive group, solid
support and carrier molecule. In another embodiment, the rhodamine
and rhodol fluorophores disclosed herein comprise both substituted
and unsubstituted moieties on the carbon atom of the central ring
of the xanthene by substituents typically found in the
xanthene-based dyes such as phenyl and substituted-phenyl moieties.
Most preferred dyes are rhodamine, rhodol, and derivatives thereof.
The choice of the fluorophore attached to the aniline moiety will
determine the pH-sensitive compound's absorption and fluorescence
emission properties as well as its live cell properties, i.e.
ability to localize within a cell.
In certain embodiments, the fluorophore (e.g., rhodamine or rhodol)
is attached to the aniline moiety via a linker. In certain
embodiments, the fluorophore (and reactive group, carrier
molecules, and solid support) comprise a linker that is used to
covalently attach the substituents to the aniline moieties
disclosed herein. The fluorophore (and solid support, carrier
molecule or reactive group) may be directly attached to the
moieties (where the linker is a single bond) or attached through a
series of stable bonds. Preferably the fluorophore is directly
attached by a single covalent bond to the aniline moiety, but may
also be attached via a linker as described below for reactive
group, carrier molecules, and solid support. When the linker is a
series of stable covalent bonds the linker typically incorporates
1-30 nonhydrogen atoms selected from the group consisting of C, N,
O, S and P. When the linker is not a single covalent bond, the
linker may be any combination of stable chemical bonds, optionally
including, single, double, triple or aromatic carbon-carbon bonds,
as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds,
carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds,
phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and
nitrogen-platinum bonds. Typically the linker incorporates less
than 15 nonhydrogen atoms and are composed of any combination of
ether, thioether, thiourea, amine, ester, carboxamide, sulfonamide,
hydrazide bonds and aromatic or heteroaromatic bonds. Typically the
linker is a combination of single carbon-carbon bonds and
carboxamide, sulfonamide or thioether bonds. The bonds of the
linker typically result in the following moieties that may be found
in the linker ether, thioether, carboxamide, thiourea, sulfonamide,
urea, urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy,
cycloalkyl and amine moieties. Examples of a linker include, but
are not limited to, substituted or unsubstituted polymethylene,
arylene, alkylarylene, arylenealkyl, or arylthio.
In certain embodiments, the linker contains 1-6 carbon atoms. In
certain embodiments, the linker comprises a thioether linkage.
Exemplary linking members include a moiety that includes, but is
not limited to, --C(O)NH--, --C(O)O--, --NH--, --S--, --O--, and
the like. In certain embodiments, the linker is or incorporates the
formula --(CH.sub.2).sub.d(CONH(CH.sub.2).sub.e).sub.z--, or where
d is an integer from 0 to 5, e is an integer from 1 to 5 and z is 0
or 1. In certain embodiments, the linker is or incorporates the
formula --O(CH.sub.2)--. In certain embodiments, the linker is or
incorporates a phenylene or a 2-carboxy-substituted phenylene.
Any combination of linkers may be used to attach the carrier
molecule, solid support or reactive group and the present
pH-sensitive fluorescent dye compounds together. The linker may
also be substituted to alter the physical properties of the
fluorophore or aniline moiety, such as spectral properties of the
pH-sensitive fluorescent dye compounds.
Another important feature of the linker is to provide an adequate
space between the carrier molecule, reactive group or solid support
and the aniline moiety or fluorophore so as to prevent steric
hindrance. Therefore, the linker of the pH-sensitive fluorescent
dye compounds disclosed herein is important for (1) attaching the
carrier molecule, reactive group or solid support to the dye
compounds and attaching the fluorophore to the aniline moiety, (2)
providing an adequate space between the carrier molecule, reactive
group or solid support and the dye compound so as not to sterically
hinder the action of the compound and (3) for altering the physical
properties of the dye compounds disclosed herein.
The pH sensing or electron rich aniline moiety of the pH-sensitive
fluorescent dye compounds disclosed herein is any moiety that, when
protonated, results in the compound being fluorescent, whilst the
dye compound is quenched when the aniline moiety is not in the
protonated state. The pH-sensitive fluorescent dye compounds often
have a pKa value in the range of about 2 to about 10. In certain
embodiments the pKa of the pH-sensitive fluorescent dye compound is
about 3 to about 10. In certain embodiments, the pKa of the
pH-sensitive fluorescent dye compound is about 5 to about 8. In
certain embodiments the pKa of the pH-sensitive fluorescent dye
compound is about 6 to about 8. In certain embodiments the pKa of
the pH-sensitive fluorescent dye compound is about 6 to about 7. In
certain embodiment the pKa of the pH-sensitive fluorescent dye
compound is about 6.5. Preferably the pKa of the pH-sensitive
fluorescent dye compounds provided herein is about 6 to about
7.
To tune the pKa to about 6 to about 7, electron donating groups
(EDG) were introduced into the aniline moiety on the aryl group.
This combined with the presence of an alkoxy or other like
substituents on the aryl when a OH or SH were not present,
unexpectedly resulted in pH-sensitive fluorescent dye compounds
that were stable in an aqueous environment and provided a pKa in
the desired range. As disclosed in and with reference to the
formulae herein, the amino group of the aniline moiety may be
substituted or replaced by another basic moiety of higher pKa.
Advantageously, the addition of a dialkylamino group, wherein each
of the alkyl groups, which may be the same or different, is
independently alkyl or substituted alkyl, to the aniline group at
position R.sup.3 resulted in the unexpected advantage of yielding
pKa values in the physiological range. In certain embodiments, the
dialkyl amino group is dimethylamino. In certain embodiments, the
dialkylamino group is diethylamino.
In certain embodiments, without wishing to be bound by a theory,
the functioning of the pH-sensitive fluorescent dye compounds
provided herein as a pH indicator is illustrated below in Scheme
2:
##STR00012##
wherein X is a fluorophore and R.sup.4 and R.sup.5 are as
previously described herein.
The EDG is typically at R.sup.3, but may be located at any position
on the aryl group. Electron donating groups of the present
invention include, for example, alkoxy, substituted alkoxy, amino,
substituted amino, dialkylamino, halogen, alkylthio, acylamino, and
(carboxyl ester)oxy. Preferred EDGs are --OCH.sub.3, --NH.sub.2,
--NHCH.sub.3, --N(CH.sub.3).sub.2, --N(CH.sub.2CH.sub.3).sub.2,
sulfotetraflurophenol (STP) ester, sulfordichlorophenol (SDP)
ester, succinimidyl (SE) ester and tetrafluorophenol (TFP) ester.
In certain embodiments, Z is O-alkyl. In certain embodiments, Z is
thioalkyl.
In certain embodiments novel dye compounds are provided for use as
fluorescent pH sensors, the dye compounds having structural formula
(I):
##STR00013## wherein
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl or substituted alkyl;
R.sup.4 is selected from the group consisting of alkyl and
substituted alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance;
R.sup.a is H, alkyl, or substituted alkyl;
R.sup.b is alkyl or substituted alkyl; and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.10 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H or halogen;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.10 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H, Cl or F;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.10 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6 are each H;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.10 are as follows:
R.sup.1 is methoxy; R.sup.2 and R.sup.6 are each H;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently methyl or ethyl;
R.sup.4 is methyl or ethyl;
R.sup.5 is methyl; ethyl; carboxyalkyl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c; wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c; and
R.sup.7, R.sup.8, R.sup.9, and R.sup.10, which may be the same or
different, are each independently methyl or propenyl.
In certain embodiments, novel dye compounds are provided for use as
fluorescent pH sensors, the dye compounds having structural formula
(II):
##STR00014## wherein
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H, halogen, --OR.sup.a, --SR.sup.a,
--NR.sup.aR.sup.b, or an electron donating group;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl or substituted alkyl;
R.sup.4 is selected from the group consisting of alkyl and
substituted alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c,
wherein L is a linker, R.sub.x is a reactive group, and S.sub.c is
a conjugated substance;
R.sup.a is H, alkyl, or substituted alkyl;
R.sup.b is alkyl or substituted alkyl; and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.8 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H or halogen;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.8 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6, which may be the same or different, are each
independently H, Cl or F;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.8 are as follows:
R.sup.1 is alkoxy or thioalkyl;
R.sup.2 and R.sup.6 are each H;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently alkyl;
R.sup.4 is alkyl;
R.sup.5 is selected from the group consisting of alkyl; substituted
alkyl; alkenyl; substituted alkenyl; acyl; aryl; substituted aryl;
carboxyalkyl; heteroaryl; substituted heteroaryl; heterocyclyl;
substituted heterocyclyl; alkylcarboxy; alkylalkoxycarbonyl;
alkylaminocarbonyl; alkylaryloxycarbonyl; alkylheteroaryl;
(CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c, wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group; -L-R.sub.x; and -L-S.sub.c;
and
R.sup.7 and R.sup.8, which may be the same or different, are each
independently alkyl or alkenyl.
In certain embodiments, R.sup.1-R.sup.8 are as follows:
R.sup.1 is methoxy;
R.sup.2 and R.sup.6 are each H;
R.sup.3 is --NR'R'', wherein R' and R'', which may be the same or
different, are each independently methyl or ethyl;
R.sup.4 is methyl or ethyl;
R.sup.5 is methyl; ethyl; carboxyalkyl; (CH.sub.2).sub.nCO(O)R;
(CH.sub.2).sub.nC(O)R; (CH.sub.2).sub.nC(O)NHR;
(CH.sub.2).sub.nC(O)NRR.sup.c; wherein n is an integer from 1 to 6,
and R and R.sup.c, which may be the same or different, are each
independently H, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, substituted amino,
alkylaminocarbonyl, aminodextran, amide, a protein, a lipophilic
group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c;
R.sup.7 and R.sup.8, which may be the same or different, are each
independently methyl or propenyl.
In certain embodiments, the EDG is selected from the group
consisting of alkoxy, substituted alkoxy, amino, substituted amino,
halogen, alkylthio, acylamino, and (carboxyl ester)oxy. In certain
embodiments, the EDG is not hydroxy or thiol. In certain
embodiments, the EDG is a dialkylamino group. In certain
embodiments, the dialkylamino group is dimethylamino or
diethylamino.
In certain embodiments, R.sup.1 is --OCH.sub.3 and R.sup.3 is
--N(CH.sub.3).sub.2 or --N(CH.sub.2CH.sub.3).sub.2.
In certain embodiments, R.sup.4 and R.sup.5 are alkyl or
substituted alkyl. In certain embodiments, R.sup.5 is methyl;
ethyl; carboxyalkyl; (CH.sub.2).sub.nCO(O)R; (CH.sub.2).sub.nC(O)R;
(CH.sub.2).sub.nC(O)NHR; (CH.sub.2).sub.nC(O)NRR.sup.c; wherein n
is an integer from 1 to 6, and R and R.sup.c, which may be the same
or different, are each independently H, alkyl, substituted alkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl,
substituted amino, alkylaminocarbonyl, aminodextran, amide, a
protein, a lipophilic group, or R.sup.d, wherein R.sup.d is
(CH.sub.2).sub.nC(O)NHR.sub.x, wherein n is an integer from 1 to 6,
and R.sub.x is a reactive group selected from carboxyl,
carboxylester, amide, maleimide, succinimidyl ester (SE),
sulfodichlorophenol (SDP) ester, sulfotetrafluorophenol (STP)
ester, tetrafluorophenol (TFP) ester, acetoxymethoxy (AM) ester,
nitrilotriacetic acid (NTA), aminodextran, DIBO-amine; -L-R.sub.x;
or -L-S.sub.c.
In certain embodiments of any of the previous embodiments, the pKa
of the pH-sensitive fluorescent dye compound is about 5 to about 8.
In certain embodiments, the pKa of the pH-sensitive fluorescent dye
compound is about 6 to about 8. In certain embodiments, the pKa of
the pH-sensitive fluorescent dye compound is about 6 to about 7. In
certain embodiments, the pKa of the pH-sensitive fluorescent dye
compound is about 6.5. In certain embodiments, the pKa of the
pH-sensitive fluorescent dye compound is about 3 to about 10.
The 4-position nitrogen of the aniline or aniline-like ring of the
compounds disclosed herein does of course always have a permissible
valency.
Reactive Groups:
In certain embodiments, the pH-sensitive fluorescent dye compounds
provided herein are chemically reactive, and are substituted by at
least one reactive group (R.sub.x). The reactive group functions as
the site of attachment for another moiety, such as a carrier
molecule or a solid support, wherein the reactive group chemically
reacts with an appropriate reactive or functional group on the
carrier molecule or solid support. Thus, in certain embodiments,
the pH-sensitive fluorescent dye compounds provided herein comprise
an aniline moiety, linker, fluorophore, a reactive group moiety and
optionally a carrier molecule and/or a solid support.
In certain embodiments, the pH-sensitive fluorescent dye compounds
provided herein further comprise a reactive group which is a member
selected from an acrylamide, an activated ester of a carboxylic
acid, a carboxylic ester, an acyl azide, an acyl nitrile, an
aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an
aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a
haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido
ester, an isocyanate, an isothiocyanate, a maleimide, a
phosphoramidite, a photoactivatable group, a reactive platinum
complex, a silyl halide, a sulfonyl halide, and a thiol. In certain
embodiments the reactive group is selected from the group
consisting of carboxylic acid, succinimidyl ester of a carboxylic
acid, hydrazide, amine and a maleimide. The reactive group may be
attached to any appropriate site on the reporter molecule or the
aniline moiety. In certain embodiments, at least one member
selected from R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is a reactive group. Preferably, at least one of R.sup.3,
R.sup.4, R.sup.5, and R.sup.6 is a reactive group, most preferred
is at least one of R.sup.4 or R.sup.5. Alternatively, if the
pH-sensitive fluorescent dye compounds disclosed herein comprise a
carrier molecule or solid support a reactive group may be
covalently attached independently to those substituents, allowing
for further conjugation to a fluorophore, carrier molecule or solid
support.
These reactive groups are synthesized during the formation of the
pH-sensitive fluorescent dye compounds provided herein and carrier
molecule- and/or solid support-containing compounds to provide
chemically reactive pH-sensitive fluorescent dye compounds. In this
way, pH-sensitive fluorescent dye compounds incorporating a
reactive group may be covalently attached to a wide variety of
carrier molecules or solid supports that contain, or are modified
to contain, functional groups with suitable reactivity, resulting
in chemical attachment of the components. In certain embodiments,
the reactive group of the pH-sensitive fluorescent dye compounds
disclosed herein and the functional group of the carrier molecule
or solid support comprise electrophiles and nucleophiles that can
generate a covalent linkage between them. In certain embodiments,
the reactive group comprises a photoactivatable group, which
becomes chemically reactive only after illumination with light of
an appropriate wavelength. Typically, the conjugation reaction
between the reactive group and the carrier molecule or solid
support results in one or more atoms of the reactive group being
incorporated into a new linkage attaching the pH-sensitive
fluorescent dye compounds disclosed herein to the carrier molecule
or solid support. Selected examples of functional groups and
linkages are shown in Table 2, where the reaction of an
electrophilic group and a nucleophilic group yields a covalent
linkage.
TABLE-US-00002 TABLE 2 Examples of some routes to useful covalent
linkages Electrophilic Group Nucleophilic Group Resulting Covalent
Linkage activated esters* amines/anilines carboxamides acrylamides
thiols thioethers acyl azides** amines/anilines carboxamides acyl
halides amines/anilines carboxamides acyl halides alcohols/phenols
esters acyl nitriles alcohols/phenols esters acyl nitriles
amines/anilines carboxamides aldehydes amines/anilines imines
aldehydes or ketones hydrazines hydrazones aldehydes or ketones
hydroxylamines oximes alkyl halides amines/anilines alkyl amines
alkyl halides carboxylic acids esters alkyl halides thiols
thioethers alkyl halides alcohols/phenols ethers alkyl sulfonates
thiols thioethers alkyl sulfonates carboxylic acids esters alkyl
sulfonates alcohols/phenols ethers anhydrides alcohols/phenols
esters anhydrides amines/anilines carboxamides aryl halides thiols
thiophenols aryl halides amines aryl amines aziridines thiols
thioethers boronates glycols boronate esters carbodiimides
carboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic
acids esters epoxides thiols thioethers haloacetamides thiols
thioethers haloplatinate amino platinum complex haloplatinate
heterocycle platinum complex haloplatinate thiol platinum complex
halotriazines amines/anilines aminotriazines halotriazines
alcohols/phenols triazinyl ethers halotriazines thiols triazinyl
thioethers imido esters amines/anilines amidines isocyanates
amines/anilines ureas isocyanates alcohols/phenols urethanes
isothiocyanates amines/anilines thioureas maleimides thiols
thioethers phosphoramidites alcohols phosphite esters silyl halides
alcohols silyl ethers sulfonate esters amines/anilines alkyl amines
sulfonate esters thiols thioethers sulfonate esters carboxylic
acids esters sulfonate esters alcohols ethers sulfonyl halides
amines/anilines sulfonamides sulfonyl halides phenols/alcohols
sulfonate esters *Activated esters, as understood in the art,
generally have the formula --CO.OMEGA., where .OMEGA. is a suitable
leaving group (e.g., succinimidyloxy (--OC.sub.4H.sub.4O.sub.2)
sulfosuccinimldyloxy (--OC.sub.4H.sub.3O.sub.2--SO.sub.3H),
-1-oxybenzotriazolyl (--OC.sub.6H.sub.4N.sub.3); or an aryloxy
group or aryloxy substituted one or more times by electron
withdrawing substituents such as nitro, fluoro, chloro, cyano, or
trifluoromethyl, or combinations thereof, used to form activated
aryl esters; or a carboxylic acid activated by a carbodiimide to
form an anhydride or mixed anhydride --OCOR.sup.x or
--NR.sup.xNHR.sup.y, where R.sup.x and R.sup.y, which may be the
same or different, are C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
perfluoroalkyl, or C.sub.1-C.sub.6 alkoxy; or cyclohexyl,
3-dimethylaminopropyl, or N-morpholinoethyl). **Acyl azides can
also rearrange to isocyanates.
The choice of the reactive group used to attach the pH-sensitive
fluorescent dye compounds disclosed herein to the substance to be
conjugated typically depends on the reactive or functional group on
the substance to be conjugated and the type or length of covalent
linkage desired. The types of functional groups typically present
on the organic or inorganic substances (biomolecule or
non-biomolecule) include, but are not limited to, amines, amides,
thiols, alcohols, phenols, aldehydes, ketones, phosphates,
imidazoles, hydrazines, hydroxylamines, disubstituted amines,
halides, epoxides, silyl halides, carboxylate esters, sulfonate
esters, purines, pyrimidines, carboxylic acids, olefinic bonds, or
a combination of these groups. A single type of reactive site may
be available on the substance (typical for polysaccharides or
silica), or a variety of sites may occur (e.g., amines, thiols,
alcohols, phenols), as is typical for proteins.
Typically, the reactive group will react with an amine, a thiol, an
alcohol, an aldehyde, a ketone, or with silica. Preferably,
reactive groups react with an amine or a thiol functional group, or
with silica. In certain embodiments, the reactive group is an
acrylamide, an activated ester of a carboxylic acid, an acyl azide,
an acyl nitrile, an aldehyde, an alkyl halide, a silyl halide, an
anhydride, an aniline, an aryl halide, an azide, an aziridine, a
boronate, a diazoalkane, a haloacetamide, a halotriazine, a
hydrazine (including hydrazides), an imido ester, an isocyanate, an
isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum
complex, a sulfonyl halide, or a thiol group. As used herein,
"reactive platinum complex" refers to chemically reactive platinum
complexes such as described in U.S. Pat. No. 5,714,327, herein
incorporated by reference in its entirety.
In certain embodiments, the pH-sensitive fluorescent dye compounds
disclosed herein comprise at least one reactive group that
selectively reacts with an amine group. This amine-reactive group
is selected from the group consisting of succinimidyl ester (SE),
sulfonyl halide, tetrafluorophenyl (TFP) ester, sulfodichlorophenol
(SDP) ester, sulfotetrafluorophenol (STP) ester, acetoxymethoxy
(AM) ester, nitrilotriacetic acid (NTA), aminodextran, DIBO-amine
and iosothiocyanates. Thus, in certain embodiments, the
pH-sensitive fluorescent dye compounds provided herein form a
covalent bond with an amine containing molecule in a sample. In
certain embodiments, the pH-sensitive fluorescent dye compounds
provided herein comprise at least one reactive group that
selectively reacts with a thiol group. This thiol-reactive group is
selected from the group consisting of maleimide, haloalkyl and
haloacetamide (including any reactive groups disclosed in U.S. Pat.
Nos. 5,362,628; 5,352,803 and 5,573,904, all of which are herein
incorporated by reference in their entirety).
Where the reactive group is an activated ester of a carboxylic
acid, such as a succinimidyl ester of a carboxylic acid, a sulfonyl
halide, a tetrafluorophenyl (TFP) ester, a sulfodichlorophenol
(SDP) ester, a sulfotetrafluorophenol (STP) ester, an
acetoxymethoxy (AM) ester, a nitrilotriacetic acid (NTA), an
aminodextran, a DIBO-amine or an isothiocyanate, the resulting
pH-sensitive fluorescent dye compound is particularly useful for
preparing conjugates of carrier molecules such as proteins,
nucleotides, oligonucleotides, or haptens. Where the reactive group
is a maleimide, haloalkyl or haloacetamide (including any reactive
groups disclosed in U.S. Pat. Nos. 5,362,628; 5,352,803 and
5,573,904, all of which are herein incorporated by reference in
their entirety) the resulting compound is particularly useful for
conjugation to thiol-containing substances. Where the reactive
group is a hydrazide, the resulting compound is particularly useful
for conjugation to periodate-oxidized carbohydrates and
glycoproteins, and in addition is an aldehyde-fixable polar tracer
for cell microinjection. Where the reactive group is a silyl
halide, the resulting compound is particularly useful for
conjugation to silica surfaces, particularly where the silica
surface is incorporated into a fiber optic probe subsequently used
for remote ion detection or quantitation.
In a certain embodiments, the reactive group is a photoactivatable
group such that the group is only converted to a reactive species
after illumination with an appropriate wavelength. An appropriate
wavelength is generally a UV wavelength that is less than 400 nm.
This method provides for specific attachment to only the target
molecules, either in solution or immobilized on a solid or
semi-solid matrix. Photoactivatable reactive groups include,
without limitation, benzophenones, aryl azides and diazirines.
Preferably, the reactive group is a photoactivatable group,
succinimidyl ester of a carboxylic acid, a haloacetamide,
haloalkyl, a hydrazine, an isothiocyanate, a maleimide group, an
aliphatic amine, a silyl halide, a cadaverine or a psoralen. More
preferably, the reactive group is a succinimidyl ester of a
carboxylic acid, a maleimide, an iodoacetamide, or a silyl halide.
In certain embodiments, the reactive group is a succinimidyl ester
of a carboxylic acid, a sulfonyl halide, a tetrafluorophenyl ester,
an iosothiocyanates or a maleimide. In certain embodiments, the
reactive group is selected from sulfodichlorophenyl (SDP) ester,
sulfotetrafluorophenol (STP) ester, succinimidyl (SE) ester and
tetrafluorophenol (TFP) ester.
Carrier Molecules:
In certain embodiments, the pH-sensitive fluorescent dye compounds
provided herein are covalently bound to a carrier molecule. If the
pH-sensitive fluorescent dye compound has a reactive group, then
the carrier molecule can alternatively be linked to the
pH-sensitive fluorescent dye compound through the reactive group.
The reactive group may contain both a reactive functional moiety
and a linker, or only the reactive functional moiety.
A variety of carrier molecules are useful herein. Exemplary carrier
molecules include antigens, steroids, vitamins, drugs, haptens,
metabolites, toxins, environmental pollutants, amino acids,
peptides, proteins, nucleic acids, nucleic acid polymers,
carbohydrates, lipids, polymers and bacterial particles. In certain
embodiments, at least one member selected from R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is a carrier molecule.
Preferably, at least one of R.sup.3, R.sup.4, R.sup.5, and R.sup.6
is a carrier molecule, most preferred is at least one of R.sup.4 or
R.sup.5.
In certain embodiments, the carrier molecule comprises an amino
acid, a peptide, a protein, a polysaccharide, a nucleoside, a
nucleotide, an oligonucleotide, a nucleic acid, a hapten, a
psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic
polymer, a polymeric microparticle, a biological cell, a virus and
combinations thereof. In certain embodiments, the carrier molecule
is selected from a hapten, a nucleotide, an oligonucleotide, a
nucleic acid polymer, a protein, a peptide or a polysaccharide. In
certain embodiments the carrier molecule is amino acid, a peptide,
a protein, a polysaccharide, a nucleoside, a nucleotide, an
oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a
hormone, a lipid, a lipid assembly, a tyramine, a synthetic
polymer, a polymeric microparticle, a biological cell, cellular
components, an ion chelating moiety, an enzymatic substrate or a
virus. In certain embodiments, the carrier molecule is an antibody
or fragment thereof, an antigen, an avidin or streptavidin, a
biotin, a dextran, an IgG binding protein, a fluorescent protein,
agarose, and a non-biological microparticle. In certain
embodiments, carrier molecules may comprise a label or a
fluorescent dye or quencher.
In certain embodiments, the carrier molecule is an amino acid
(including those that are protected or are substituted by
phosphates, carbohydrates, or C.sub.1 to C.sub.22 carboxylic
acids), or a polymer of amino acids such as a peptide or protein.
In certain embodiments, the carrier molecule contains at least five
amino acids, more preferably 5 to 36 amino acids. Exemplary
peptides include, but are not limited to, neuropeptides, cytokines,
toxins, protease substrates, and protein kinase substrates. Other
exemplary peptides may function as organelle localization peptides,
that is, peptides that serve to target the conjugated compound for
localization within a particular cellular substructure by cellular
transport mechanisms. Preferred protein carrier molecules include
enzymes, antibodies, lectins, glycoproteins, histones, albumins,
lipoproteins, avidin, streptavidin, protein A, protein G,
phycobiliproteins and other fluorescent proteins, hormones, toxins
and growth factors. Typically, the protein carrier molecule is an
antibody, an antibody fragment, avidin, streptavidin, a toxin, a
lectin, a growth factor, bacterial particle or a binding partner
for a cell receptor.
In certain embodiments, the carrier molecule comprises a nucleic
acid base, nucleoside, nucleotide or a nucleic acid polymer,
optionally containing an additional linker or spacer for attachment
of a fluorophore or other ligand, such as an alkynyl linkage (U.S.
Pat. No. 5,047,519), an aminoallyl linkage (U.S. Pat. No.
4,711,955) or other linkage. In certain embodiments, the nucleotide
carrier molecule is a nucleoside or a deoxynucleoside or a
dideoxynucleoside.
Exemplary nucleic acid polymer carrier molecules are single- or
multi-stranded, natural or synthetic DNA or RNA oligonucleotides,
or DNA/RNA hybrids, or incorporating an unusual linker such as
morpholine derivatized phosphates (AntiVirals, Inc., Corvallis
Oreg.), or peptide nucleic acids such as N-(2-aminoethyl)glycine
units, where the nucleic acid contains fewer than 50 nucleotides,
more typically fewer than 25 nucleotides.
In certain embodiments, the carrier molecule comprises a
carbohydrate or polyol that is typically a polysaccharide, such as
dextran, FICOLL.RTM., heparin, glycogen, amylopectin, mannan,
inulin, starch, agarose and cellulose, or is a polymer such as a
poly(ethylene glycol). In certain embodiments, the polysaccharide
carrier molecule includes dextran, agarose or FICOLL.RTM..
In certain embodiments, the carrier molecule comprises a lipid
(typically having 6-25 carbons), including glycolipids,
phospholipids, and sphingolipids. In certain embodiments, the
carrier molecule comprises a lipid vesicle, such as a liposome, or
is a lipoprotein. Some lipophilic substituents are useful for
facilitating transport of the conjugated dye into cells or cellular
organelles.
In certain embodiments, the carrier molecule is a cell, cellular
system, cellular fragment, or subcellular particles, including
virus particles, bacterial particles, virus components, biological
cells (such as animal cells, plant cells, bacteria, or yeast), or
cellular components. Examples of cellular components that are
useful as carrier molecules include lysosomes, endosomes,
cytoplasm, nuclei, histones, mitochondria, Golgi apparatus,
endoplasmic reticulum and vacuoles.
In certain embodiments, embodiment, the carrier molecule
non-covalently associates with organic or inorganic materials.
Exemplary embodiments of the carrier molecule that possess a
lipophilic substituent may be used to target lipid assemblies such
as biological membranes or liposomes by non-covalent incorporation
of the pH-sensitive fluorescent dye compound within the membrane,
e.g., for use as probes for membrane structure or for incorporation
in liposomes, lipoproteins, films, plastics, lipophilic
microspheres or similar materials.
In certain embodiments, the carrier molecule comprises a specific
binding pair member wherein the pH-sensitive fluorescent dye
compounds provided herein are conjugated to a specific binding pair
member and used to the formation of the bound pair. Alternatively,
the presence of the labeled specific binding pair member indicates
the location of the complementary member of that specific binding
pair; each specific binding pair member having an area on the
surface or in a cavity which specifically binds to, and is
complementary with, a particular spatial and polar organization of
the other. In this instance, the dye compounds disclosed herein
function as a reporter molecule for the specific binding pair.
Exemplary binding pairs are set forth in Table 3.
TABLE-US-00003 TABLE 3 Representative Specific Binding Pairs
Antigen Antibody biotin avidin (or streptavidin or anti-biotin)
IgG* protein A or protein G drug drug receptor folate folate
binding protein toxin toxin receptor carbohydrate lectin or
carbohydrate receptor peptide peptide receptor protein protein
receptor enzyme substrate enzyme DNA (RNA) cDNA (cRNA).sup..dagger.
hormone hormone receptor ion chelator *IgG is an immunoglobulin
.sup..dagger.cDNA and cRNA are the complementary strands used for
hybridization
Solid Supports:
In certain embodiments, the pH-sensitive dye compounds disclosed
herein are covalently bonded to a solid support. The solid support
may be attached to the dye compounds either through the aniline
moiety, fluorophore, or through a reactive group, if present, or
through a carrier molecule, if present. Even if a reactive group
and/or a carrier molecule are present, the solid support may be
attached through the aniline moiety or fluorophore. In certain
embodiments, at least one member selected from R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is a solid support.
Preferably, at least one of R.sup.3, R.sup.4, R.sup.5, and R.sup.6
is a solid support, most preferred is at least one of R.sup.4 or
R.sup.5.
Solid supports suitable for use herein are typically substantially
insoluble in liquid phases. Solid supports for use herein are not
limited to a specific type of support. Rather, a large number of
supports are available and are known to one of ordinary skill in
the art. Thus, useful solid supports include solid and semi-solid
matrixes, such as aerogels and hydrogels, resins, beads, biochips
(including thin film coated biochips), microfluidic chip, a silicon
chip, multi-well plates (also referred to as microtitre plates or
microplates), membranes, conducting and nonconducting metals, glass
(including microscope slides) and magnetic supports. More specific
examples of useful solid supports include silica gels, polymeric
membranes, particles, derivatized plastic films, glass beads,
cotton, plastic beads, alumina gels, polysaccharides such as
Sepharose.RTM., poly(acrylate), polystyrene, poly(acrylamide),
polyol, agarose, agar, cellulose, dextran, starch, FICOLL.RTM.,
heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose,
diazocellulose, polyvinylchloride, polypropylene, polyethylene
(including poly(ethylene glycol)), nylon, latex bead, magnetic
bead, paramagnetic bead, superparamagnetic bead, starch and the
like.
In certain embodiments, the solid support may include a solid
support reactive functional group, including, but not limited to,
hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano,
amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate,
sulfonamide, sulfoxide, etc., for attaching the dye compounds
disclosed herein. Useful reactive groups are disclosed above and
are equally applicable to the solid support reactive functional
groups herein.
A suitable solid phase support may be selected on the basis of
desired end use and suitability for various synthetic protocols.
For example, where amide bond formation is desirable to attach the
pH-sensitive fluorescent dye compounds disclosed herein to the
solid support, resins generally useful in peptide synthesis may be
employed, such as polystyrene (e.g., PAM-resin obtained from Bachem
Inc., Peninsula Laboratories, etc.), POLYHIPE.TM. resin (obtained
from Aminotech, Canada), polyamide resin (obtained from Peninsula
Laboratories), polystyrene resin grafted with polyethylene glycol
(TentaGel.TM., Rapp Polymere, Tubingen, Germany),
polydimethyl-acrylamide resin (available from Milligen/Biosearch,
California), or PEGA beads (obtained from Polymer
Laboratories).
Preparation of Conjugates:
In certain embodiments, conjugates of the pH-sensitive fluorescent
dye compounds disclosed herein are provided. Conjugates of
components (carrier molecules or solid supports), e.g., drugs,
peptides, toxins, nucleotides, phospholipids, proteins and other
organic molecules are prepared by organic synthesis methods using
the pH-sensitive fluorescent dye compounds disclosed herein, are
generally prepared by means well recognized in the art (Haugland,
MOLECULAR PROBES HANDBOOK, supra, (2002)). Preferably, conjugation
to form a covalent bond consists of mixing the reactive dye
compounds disclosed herein in a suitable solvent in which both the
pH-sensitive fluorescent dye compound and the substance to be
conjugated are soluble. The reaction preferably proceeds
spontaneously without added reagents at room temperature or below.
For those reactive compounds that are photoactivated, conjugation
is facilitated by illumination of the reaction mixture to activate
the reactive compound. Chemical modification of water-insoluble
substances, so that a desired compound-conjugate may be prepared,
is preferably performed in an aprotic solvent such as
dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate,
toluene, or chloroform. Similar modification of water-soluble
materials is readily accomplished through the use of the instant
reactive compounds to make them more readily soluble in organic
solvents.
Preparation of peptide or protein conjugates typically comprises
first dissolving the protein to be conjugated in aqueous buffer at
about 1-10 mg/mL at room temperature or below. Bicarbonate buffers
(pH about 8.3) are especially suitable for reaction with
succinimidyl esters, phosphate buffers (pH about 7.2 to about 8)
for reaction with thiol-reactive functional groups and carbonate or
borate buffers (pH about 9) for reaction with isothiocyanates and
dichlorotriazines. The appropriate reactive compound is then
dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in an
amount sufficient to give a suitable degree of conjugation when
added to a solution of the protein to be conjugated. The
appropriate amount of compound for any protein or other component
is conveniently predetermined by experimentation in which variable
amounts of the compound are added to the protein, the conjugate is
chromatographically purified to separate unconjugated compound and
the compound-protein conjugate is tested in its desired
application.
Following addition of the pH-sensitive fluorescent dye compound to
the component solution, the mixture is incubated for a suitable
period (typically about 1 hour at room temperature to several hours
on ice), the excess pH-sensitive fluorescent dye compound is
removed by gel filtration, dialysis, HPLC, adsorption on an ion
exchange or hydrophobic polymer or other suitable means. The
pH-sensitive fluorescent dye compound-conjugate may be used in
solution or lyophilized. In this way, suitable conjugates may be
prepared from antibodies, antibody fragments, avidins, lectins,
enzymes, proteins A and G, cellular proteins, albumins, histones,
growth factors, hormones, and other proteins.
Conjugates of polymers, including biopolymers and other higher
molecular weight polymers are typically prepared by means well
recognized in the art (for example, Brinkley et al., Bioconjugate
Chem., 3:2 (1992)). In these embodiments, a single type of reactive
site may be available, as is typical for polysaccharides) or
multiple types of reactive sites (e.g. amines, thiols, alcohols,
phenols) may be available, as is typical for proteins. Selectivity
of labeling is best obtained by selection of an appropriate
reactive dye compound. For example, modification of thiols with a
thiol-selective reagent such as a haloacetamide or maleimide, or
modification of amines with an amine-reactive reagent such as an
activated ester, acyl azide, isothiocyanate or
3,5-dichloro-2,4,6-triazine. Partial selectivity can also be
obtained by careful control of the reaction conditions.
When modifying polymers with the pH-sensitive fluorescent dye
compounds disclosed herein, an excess of pH-sensitive fluorescent
dye compound is typically used, relative to the expected degree of
pH-sensitive fluorescent dye compound substitution. Any residual,
unreacted pH-sensitive fluorescent dye compound or a pH-sensitive
fluorescent dye compound hydrolysis product is typically removed by
dialysis, chromatography or precipitation. Presence of residual,
unconjugated dye can be detected by thin layer chromatography using
a solvent that elutes the dye away from its conjugate. In all cases
it is usually preferred that the reagents be kept as concentrated
as practical so as to obtain adequate rates of conjugation.
In certain embodiments, the conjugates disclosed herein are
associated with an additional substance, that binds either to the
fluorophore or the conjugated substance (carrier molecule or solid
support) through noncovalent interaction. In another exemplary
embodiment, the additional substance is an antibody, an enzyme, a
hapten, a lectin, a receptor, an oligonucleotide, a nucleic acid, a
liposome, or a polymer. The additional substance is optionally used
to probe for the location of the dye-conjugate, for example, as a
means of enhancing the signal of the dye-conjugate.
In certain embodiments, compositions are provided for determining
the pH of a sample, the compositions comprising:
a) one or more of the pH-sensitive fluorescent dye compounds
described herein; and
b) a carrier,
wherein the one or more of the pH-sensitive fluorescent dye
compounds are present in an amount effective to detect the pH of
the sample.
In certain embodiments, compositions are provided for determining
the pH of a sample, the compositions comprising:
(a) one or more of the pH-sensitive fluorescent dye compounds
described herein; and
(b) an analyte,
wherein the one or more of the pH-sensitive fluorescent dye
compounds are present in an amount effective to detect the pH of
the sample.
In certain embodiments, the analyte is a cell and the pH-sensitive
fluorescent dye compound is located inside the cell. In certain
embodiments, the analyte is a protein, lipid or nucleic acid. In
certain embodiments, the pH-sensitive fluorescent dye compound is
conjugated to a carrier molecule.
Methods:
In certain embodiments, the pH-sensitive fluorescent dye compounds,
dye-conjugates and compositions provided herein may be used in
methods including, but not limited to, methods to determine the pH
of living cells or cell compartments, to determine a change in pH
to the local environment caused by a cell, and directly and
indirectly detect specific cellular events associated with a change
in pH. In certain embodiments, the methods involve detecting
contamination in cell culture or on agar plates. For sake of
clarity, the sample may also include material other than live cells
and cell compartments such as, but not limited to, cell culture
medium, biological fluids, diagnostic materials, and bacterial
medium such as agar plates. As used herein, the term "a cell
compartment" refers to one of the many organelles suspended in the
cell cytoplasm. The pH of a cell or cell compartment may be
measured by introducing one or more of the pH-sensitive fluorescent
dye compounds, dye conjugates or compositions provided herein into
a cell or cell compartment, irradiating the dye or dye conjugate
with a suitable light source, and observing the intensity of
fluorescence of the dye or conjugate. The observed fluorescence
intensity may then be used to determine pH by a variety of methods
known in the field, selected according to the method of
accumulation of the dye or dye conjugate. For instance, the
observed fluorescence may be compared to a known standard, for
example a calibration curve of fluorescence intensity versus pH, or
to fluorescence intensity measurements indicative of the total
pH-sensitive fluorescent dye compound, dye conjugate, or
composition present. Any conventional fluorimetric equipment may be
used to irradiate the sample, and to measure the resulting
fluorescent response.
As stated above, the sample may comprise live cells, intracellular
fluids, extracellular fluids, biological fluids, sera, biological
fermentation media, environmental sample, industrial samples,
proteins, peptides, buffer solutions, biological fluids or chemical
reactors, blood cells, immune cells, cultured cells, muscle tissue,
neurons, extracellular vesicles; vascular tissue, blood fluids,
saliva, urine, water, soil, waste water, sea water;
pharmaceuticals, foodstuffs or beverages. In certain embodiments,
the sample is immobilized on a polymeric membrane, within a
polymeric gel, on a microparticle, on a microarray, on a silicon
chip, on a glass slide, on a microwell plate, and on a microfluidic
chip.
The pH-sensitive fluorescent dye compounds disclosed herein may
therefore be used as pH sensors in relation to samples comprising
or suspected of comprising a biological entity or biological
substance. The pH-sensitive fluorescent dye compounds disclosed
herein may be used in assays involving a biological entity or
biological substance. In certain embodiments, the current teachings
provide for the use of the pH-sensitive fluorescent dye compounds
in a biological assay for the purposes described herein,
particularly as a pH sensor.
Thus, in certain embodiments, the methods disclosed herein comprise
determining the pH of a sample, wherein the methods comprise:
(a) contacting the sample with one or more of the pH-sensitive
fluorescent dye compounds disclosed herein, to form a contacted
sample;
(b) incubating the contacted sample for an appropriate amount of
time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated
sample;
wherein the fluorescent emissions are used to determine the pH of
the sample.
In certain embodiments, the methods disclosed herein comprise
determining the pH of a sample, wherein the methods comprise:
(a) contacting the sample with one or more of the compositions
provided herein to form a contacted sample;
(b) incubating the contacted sample for an appropriate amount of
time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated
sample;
wherein the fluorescent emissions are used to determine the pH of
the sample.
In certain embodiments, the pH-sensitive fluorescent dye compounds
disclosed herein are used in cell culture for detection of
contamination. In certain embodiments, the pH-sensitive fluorescent
dye compounds disclosed herein are used in or on agar plates for
the detection of contamination.
In certain embodiments, a change in the pH inside the cell
corresponds to a cellular process. In certain embodiments, the
pH-sensitive fluorescent dye compound is conjugated to a protein,
nucleic acid or lipid. In certain embodiments, the pH-sensitive
fluorescent dye compound is conjugated to transferrin. In certain
embodiments, the pH-sensitive fluorescent dye compound is
conjugated to a carrier molecule through a succinimidyl ester. In
certain embodiments the pH-sensitive fluorescent dye compound is
conjugated to epithelial growth factor (EGF) or EGF receptor
(EGFR). In certain embodiments the pH-sensitive fluorescent dye
compound is non-fluorescent before entering the cell. More
particularly, the pH-sensitive fluorescent dye compound becomes
fluorescent after entering the cell. In certain embodiments, the
pH-sensitive fluorescent dye compound enters the cell through
phagocytosis. In certain embodiments, the pH-sensitive fluorescent
dye compound enters the cell through receptor-mediated
endocytosis.
In certain embodiments, methods are provided for monitoring the pH
inside a live cell, the methods comprising:
(a) contacting the cell with one or more of the pH-sensitive
fluorescent dye compound disclosed herein to form a contacted
cell;
(b) incubating the contacted cell for a sufficient amount of time
for the one or more pH-sensitive fluorescent dye compounds to enter
the cell to form a labeled cell;
(c) illuminating the labeled cell with an appropriate wavelength to
form an illuminated cell; and
(d) detecting fluorescent emissions from the illuminated cell;
wherein the fluorescent emissions are used to monitor the pH inside
the cell.
In certain embodiments, methods are provided for monitoring the pH
inside a live cell, the methods comprising:
(a) contacting the cell with one or more of the compositions
provided herein to form a contacted cell;
(b) incubating the contacted cell for a sufficient amount of time
for the one or more compositions to enter the cell to form a
labeled cell;
(c) illuminating the labeled cell with an appropriate wavelength to
form an illuminated cell; and
(d) detecting fluorescent emissions from the illuminated cell;
wherein the fluorescent emissions are used to monitor the pH inside
the cell.
Typically, the pH-sensitive fluorescent dyes and/or dye conjugates
and/or compositions disclosed herein are introduced into a living
cell or cell compartment by mixing with a sample comprising a cell
or cell compartment, and then leaving the mixture to incubate for a
time interval adequate to allow entry of the pH-sensitive
fluorescent dye, dye conjugate, or composition into the cell or
cell compartment. During this time interval, the pH-sensitive
fluorescent dye compound, dye conjugate, or composition either
passively diffuses across the plasma membrane or is taken up by the
cell or cell compartment by a cell mediated mechanism.
In the case of conjugates, typically target molecules, including
bacterial particles that induce phagocytosis and specific binding
patterns that bind a cellular receptor and induce receptor
internalization, are generally cell or cell compartment specific,
hence a specific conjugate generally attaches to only one kind of
cell or cell compartment. Once attached to a cell or cell
compartment, the pH-sensitive fluorescent dye conjugate may diffuse
through a membrane of that cell or cell compartment or be
trafficked to a specific cell compartment by receptor-mediated
endocytosis, hence exposing itself to the internal pH of the cell
or cell compartment.
Advantageously, the pH-sensitive fluorescent dye compounds, dye
conjugates, and compositions disclosed herein allow for a more
accurate determination of pH as compared to existing pH sensor dyes
because the pKa's of the pH-sensitive fluorescent dye compounds,
dye conjugates, and compositions disclosed herein may, by design,
be adjusted by substitution to a variety of pKa values. This is
accomplished by the addition of EDG groups on the aniline moiety
and by substitution at one of the remaining R.sup.1-R.sup.6 with a
group that is not OH or SH. Thus, some are tuned to the pH of the
cell or cell compartment of interest, and consequently will be
ideal for measuring the pH of a cell or cell compartment when
accumulated by receptor-mediated endocytosis or any non-passive
accumulation mechanism as well as by passive accumulation. Others
will have a pKa far from the pH of the cell media or extracellular
matrix. The pH-sensitive fluorescent dye compounds disclosed herein
are tuned to match the sample of choice with the understanding that
the compounds become fluorescent when the pH of the sample drops
below the pKa of the pH-sensitive fluorescent dye compound(s)
disclosed herein. In certain embodiments, the pKa of the
pH-sensitive fluorescent dye compound may be modified by the
addition of a dialkyl amino group at position R.sup.3
advantageously resulting in a physiological pKa. In certain
embodiments, the dialkyl amino group is diethylamino. In certain
embodiments, the dialkylamino group is dimethylamino.
Accumulation will occur passively when one form of the pH-sensitive
fluorescent dye compound, dye conjugate, or composition with
respect to pH (the uncharged form) freely penetrates the cell or
cell compartment of interest and the other form (a charged form) is
non-penetrating. Fluorescence will approach its equilibrium
position provided the form of the accumulated dye is the
fluorescent form and that accumulation to equilibrium has occurred.
The observed fluorescence intensity may then be used to determine
pH according to any of the known methods, for instance by reference
to calibration data, or by comparing the observed fluorescence
intensity to the fluorescence intensity observed on acidifying the
test sample so that all the dye or conjugate fluoresces, the ratio
of the two fluorescence intensities coupled with the known pKa
allowing determination of pH. Passive accumulation may be achieved
by use of a pH-sensitive fluorescent dye compound that is not
attached to a carrier molecule or solid support or a pH-sensitive
fluorescent dye compound that is attached to a small, relatively
hydrophobic target molecule capable of diffusing through the cell
membrane, such as one or more acetoxymethoxy (AM) ester groups.
However, we have found that pH-sensitive fluorescent dye compounds
comprising a reactive group, such as succinimidyl ester, also
appear to passively accumulate in cells.
In certain embodiments, methods are provided for identifying a
target cell within a population of cells wherein the target cell is
differentially labeled relative to neighboring cells within the
population, the methods comprising:
(a) contacting one or more of the pH-sensitive fluorescent dye
compounds disclosed herein with the population of cells to form a
contacted cell population;
(b) incubating the contacted cell population for a period of time
sufficient for the one or more pH-sensitive fluorescent dye
compounds to enter the target cell, thereby forming an incubated
cell population; and
(c) illuminating the incubated cell population, wherein the target
cell is identified by a differential label relative to neighboring
cells within the population.
In certain embodiments, methods are provided for identifying a
target cell within a population of cells wherein the target cell is
differentially labeled relative to neighboring cells within the
population, the methods comprising:
(a) contacting one or more of the compositions disclosed herein
with the population of cells to form a contacted cell
population;
(b) incubating the contacted cell population for a period of time
sufficient for the one or more composition to enter the target
cell, thereby forming an incubated cell population; and
(c) illuminating the incubated cell population, wherein the target
cell is identified by a differential label relative to neighboring
cells within the population.
In certain embodiments, the target cell is a neuronal cell. In
certain embodiments, the neuronal cell is identified by increased
fluorescence as compared with neighboring cells. In certain
embodiments, the population of cells is part of a tissue. More
particularly, the tissue is selected from the group consisting of
tumor tissue, epidermal tissue, muscle tissue, bone marrow tissue,
neural tissue, brain tissue, organ tissue, and human biopsy
tissue.
In certain embodiments, methods are provided for identifying a
first neuron or plurality of neurons in a neural tissue slice, or a
neuronal cell is a heterogeneous mixture comprising neuronal and
non-neuronal cell types. Also provided are methods for detecting
the effect of a neuromodulator on a connection between neurons or a
plurality of neurons forming a circuit; methods for identifying an
inhibitory connection between or on neurons; and methods for
identifying neurons in vivo or in vitro.
In certain embodiments, healthy neurons are identified in mixed
cultures of living cells or preparations of cells, such as tissue
slices or whole mount. In vivo identification of neurons or other
metabolically active cells such as cardiac and skeletal myocytes
are particularly preferred methods employing the pH-sensitive dye
compounds disclosed herein.
Non-passive accumulation may occur through cell-mediated mechanism
such as phagocytosis and endocytosis, typically when a pH-sensitive
fluorescent dye compound disclosed herein comprises a carrier
molecule or solid support that is bound by a cellular receptor. In
this instance, whenever the dye compound provided herein is
accumulated in the cell or cell compartment by a mechanism that
does not rely solely on passive accumulation, the accuracy of a pH
measurement will be highest when the pKa of the dye compound is
near the pH to be measured. In this situation, without wishing to
be bound by a theory, the increased accuracy available with the
pH-sensitive dye compounds disclosed herein may arise from the fact
that the pKa is the pH of the aqueous medium containing a species
when it is 50% protonated and that at this pH a change in proton
intensity will have greatest effect on the properties of the
species. Hence, the greatest change in fluorescence intensity
occurs at the pKa of the pH-sensitive fluorescent dye, and
measurements of absolute fluorescence intensity at this pH so that
the pH-sensitive fluorescent dye compounds used to analyze a
particular cell or cell compartment embraces the pH of that cell or
cell compartment is generally sufficient.
In certain embodiments, methods are provided for detecting a pH
related intracellular process, the methods comprising:
(a) contacting one or more of the pH-sensitive fluorescent dye
compounds disclosed herein with a cell to form a contacted
cell;
(b) incubating the contacted cell to form an incubated
solution;
(c) illuminating the incubated solution to form an illuminated
solution; and
(d) detecting fluorescent emissions from the illuminated
solution;
wherein increased fluorescent emissions indicates activation of the
intracellular process.
In certain embodiments, methods are provided for detecting a pH
related intracellular process, the methods comprising:
(a) contacting one or more of the compositions provided herein with
a cell to form a contacted cell;
(b) incubating the contacted cell to form an incubated
solution;
(c) illuminating the incubated solution to form an illuminated
solution; and
(d) detecting fluorescent emissions from the illuminated
solution;
wherein increased fluorescent emissions indicates activation of the
intracellular process.
In certain embodiments, the intracellular process is the opening of
an ion channel. More particular still, the ion channel is
calcium.
In certain embodiments, the pH-sensitive fluorescent dye compound
is internalized after incubation with the cytosol of the cell.
Certain embodiments provide a no-wash, no-quench assay for
phagocytosis that is based on fluorogenic bioparticles comprising
the pH-sensitive fluorescent dye compounds provided herein. Current
protocols for measuring phagocytosis that use fluorescent
bioparticles, require a trypan blue quenching step and several
washing steps. These steps can introduce significant variability in
the assay. To address this issue, provided herein is a no-wash
phagocytosis kit, using E. coli bioparticles conjugated to a
pH-sensitive fluorescent dye as described herein. These bioparticle
conjugates are weakly fluorescent at extracellular pH. However,
when added to phagocytic J774.2 murine macrophages, they become
ingested into acidic compartments and fluoresce from within the
cells, giving specific signals that meet or exceed the brightness
of the Vybrant.TM. Phagocytosis Assay Kit (Life Technologies
Corporation). Quantitation of the phagocytic index with these
conjugates requires no wash or quenching steps, and uptake of the
bioparticles is potently inhibited by cytochalaisin D, a known
blocker of phagocytosis. The pH-sensitive fluorescent bioparticles
described herein may be used in plate based, as well as imaging and
flow cytometry assays of phagocytosis.
In certain embodiments, methods are provided for detecting
phagocytosis of a carrier molecule in solution, the methods
comprising:
(a) conjugating the carrier molecule to one or more of the
pH-sensitive fluorescent dye compounds disclosed herein to form a
carrier-dye conjugate;
(b) contacting the carrier-dye conjugate with a cell to form a
contacted cell;
(c) incubating the contacted cell to form an incubated
solution;
(d) illuminating the incubated solution to form an illuminated
solution; and
(e) detecting fluorescent emissions from the illuminated
solution;
wherein fluorescent emissions indicate phagocytosis of the carrier
molecule.
In certain embodiments, methods are provided for detecting
phagocytosis of a carrier molecule in solution, the methods
comprising:
(a) conjugating the carrier molecule to one or more of the
compositions disclosed herein to form a carrier-dye conjugate;
(b) contacting the carrier-dye conjugate with a cell to form a
contacted cell;
(c) incubating the contacted cell to form an incubated
solution;
(d) illuminating the incubated solution to form an illuminated
solution; and
(e) detecting fluorescent emissions from the illuminated
solution;
wherein fluorescent emissions indicate phagocytosis of the carrier
molecule.
In particular embodiments, the carrier molecule is an E. coli
bioparticle.
In certain embodiments, methods are provided for diagnosing or
detecting a disease in a subject, the methods comprising:
(a) contacting a sample obtained from a subject suspected of having
the disease with one or more of the pH-sensitive fluorescent dye
compounds disclosed herein, to form a contacted sample;
(b) incubating the contacted sample for an appropriate amount of
time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated
sample;
wherein the fluorescent emissions are used to diagnose or detect
the disease.
In certain embodiments, methods are provided for diagnosing or
detecting a disease in a subject, the methods comprising:
(a) contacting a sample obtained from a subject suspected of having
the disease with one or more of the compositions disclosed herein,
to form a contacted sample;
(b) incubating the contacted sample for an appropriate amount of
time to form an incubated sample;
(c) illuminating the incubated sample with an appropriate
wavelength to form an illuminated sample; and
(d) detecting fluorescent emissions from the illuminated
sample;
wherein the fluorescent emissions are used to diagnose or detect
the disease.
In certain embodiments, the disease is associated with the central
nervous system. In certain embodiments, the disease is Alzheimer's
disease (AD). In certain embodiments, the pH-sensitive fluorescent
dye compound is conjugated to a carrier molecule associated with
the disease. In certain embodiments, the pH-sensitive fluorescent
dye compound is conjugated to .beta.-amyloid or a fragment or
thereof. Accordingly, certain embodiments provides a blood based
assay for Alzheimer's disease, based on the phagocytosis of the
pH-sensitive fluorescent dye compounds disclosed herein conjugated
to .beta.-amyloid protein.
In certain embodiments, the disease is associated with the immune
system. In certain embodiments, the disease is associated with
inflammation. In certain embodiments, the disease is cancer. In
certain embodiments, the disease is associated with oxidative
stress. In certain embodiments, the pH-sensitive fluorescent dye
compound is conjugated to a carrier molecule associated with the
disease.
In certain embodiments, methods are provided for detecting a target
molecule capable of modulating a cellular process that affects the
pH or directly affects the pH of a cell. In certain embodiments,
the target molecule is a small molecule. In certain embodiments,
the cell is a neuronal cell. In certain embodiments, the cell is a
cancer cell. In certain embodiments, the cell is an immune
cell.
In certain embodiments, methods are provided for detecting any one
of the following with a pH-sensitive fluorescent dye compound as
described herein: an antibody, protein, peptide, enzyme substrate,
hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin,
avidin, streptavidin, toxin, carbohydrate, oligosaccharide,
polysaccharide, nucleic acid, derivatized deoxy nucleic acid, DNA
fragment, RNA fragment, derivatized DNA fragment, derivatized RNA
fragment, nucleoside, nucleotide, natural drug, synthetic drug,
virus particle, bacterial particle, virus component, yeast
component, blood cell, blood cell component, plasma component,
serum component, biological cell, neuronal cells, noncellular blood
component, bacteria, bacterial component, natural or synthetic
lipid vesicle, poison, environmental pollutant, polymer, polymer
particle, glass particle, glass surface, plastic particle, plastic
surface, polymer membrane, conductor or semiconductor comprising
detecting a compound disclosed herein bound to said antibody,
protein, peptide, enzyme substrate, hormone, lymphokine,
metabolite, receptor, antigen, hapten, lectin, avidin,
streptavidin, toxin, carbohydrate, oligosaccharide, polysaccharide,
nucleic acid, derivatized deoxy nucleic acid, DNA fragment, RNA
fragment, derivatized DNA fragment, derivatized RNA fragment,
nucleoside, nucleotide, natural drug, synthetic drug, virus
particle, bacterial particle, virus component, yeast component,
blood cell, blood cell component, plasma component, serum
component, biological cell, noncellular blood component, bacteria,
bacterial component, natural or synthetic lipid vesicle, poison,
environmental pollutant, polymer, polymer particle, glass particle,
glass surface, plastic particle, plastic surface, polymer membrane,
conductor or semiconductor.
In certain embodiments, methods are provided for detecting acidic
or basic conditions comprising contacting a pH-sensitive
fluorescent dye compound as described herein with a composition
suspected of being acidic or basic and detecting the fluorescence
of the pH-sensitive fluorescent dye compound as an indicator of
said acidic or basic conditions. In certain embodiments, the
composition being tested comprises an intracellular
environment.
Accuracy for the general means of measuring pH may be further
increased by using a plurality of the pH-sensitive fluorescent dye
compounds provided herein having different fluorescent responses.
In certain embodiments, two or more pH-sensitive fluorescent dye
compounds according to the present invention may be used,
optionally bonded to identical carrier molecules or solid supports,
or a pH-sensitive fluorescent dye compound as disclosed herein and
another different dye. In certain embodiments, the second
fluorescent dye has a positive fluorescence response with
increasing pH (i.e., that the intensity of fluorescence exhibited
by the dye or complex increases with increasing pH). It is
preferable that the two or more dyes have overlapping titration
ranges, and more preferably the different dyes or conjugates have
pKa values within about 1 unit of each other. The intensity of
fluorescence of each dye or conjugate is then measured, and pH
determined by calculating the ratio of the fluorescence intensity
of the first compound to the fluorescence intensity of the second
compound and comparing the value obtained to a calibration
curve.
In certain embodiments, the pH-sensitive dye compounds may be used
to analyze the kinetics of migration of a species into or through a
cell or cell compartment. This may be done by monitoring the
intensity of fluorescence of a pH-sensitive dye compound over a
time interval. Where pH is known, the pH-sensitive dye compound
should be selected so as to have a pKa in the range between the pH
at the starting point and the pH at the end point of the pathway to
be analyzed. In some cases it may be desirable to use a plurality
of pH-sensitive dye compounds having a variety of pKa values, with
each dye or complex tuned to a different portion of the pathway to
be analyzed.
In certain embodiments, methods are provided for using a
pH-sensitive fluorescent dye compound, dye conjugate, or
composition provided herein for analysis or detection. More
particularly, the detection may be performed by optical means. In
certain embodiments, the fluorescence emission is optionally
detected by visual inspection, or by use of any of the following
devices: CCD cameras, video cameras, photographic film, laser
scanning devices, fluorometers, photodiodes, quantum counters,
epifluorescence microscopes, scanning microscopes, flow cytometers,
fluorescence microplate readers, or by means for amplifying the
signal such as photomultiplier tubes.
In certain embodiments, the sample or medium in which a
pH-sensitive fluorescent dye compound provided herein is present is
illuminated with a wavelength of light selected to give a
detectable optical response, and observed with a means for
detecting the optical response. Equipment that is useful for
illuminating the pH-sensitive fluorescent dye compounds and
compositions disclosed herein includes, but is not limited to,
hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers
and laser diodes. These illumination sources are optically
integrated into laser scanners, fluorescence microplate readers or
standard or microfluorometers.
The pH-sensitive fluorescent dye compounds, dye conjugates, and
compositions disclosed herein may, at any time after or during an
assay, be illuminated with a wavelength of light that results in a
detectable optical response, and observed with a means for
detecting the optical response. Upon illumination, such as by an
ultraviolet or visible wavelength emission lamp, an arc lamp, a
laser, or even sunlight or ordinary room light, the fluorescent
compounds, including those bound to the complementary specific
binding pair member, display intense visible absorption as well as
fluorescence emission. Selected equipment that is useful for
illuminating the fluorescent pH-sensitive fluorescent dye compounds
disclosed herein include, but is not limited to, hand-held
ultraviolet lamps, mercury arc lamps, xenon lamps, argon lasers,
laser diodes, and YAG lasers. These illumination sources are
optionally integrated into laser scanners, fluorescence microplate
readers, standard or mini fluorometers, or chromatographic
detectors. This fluorescence emission is optionally detected by
visual inspection, or by use of any of the following devices: CCD
cameras, video cameras, photographic film, laser scanning devices,
fluorometers, photodiodes, quantum counters, epifluorescence
microscopes, scanning microscopes, flow cytometers, fluorescence
microplate readers, or by means for amplifying the signal such as
photomultiplier tubes. Where the sample is examined using a flow
cytometer, a fluorescence microscope or a fluorometer, the
instrument is optionally used to distinguish and discriminate
between the fluorescent compounds disclosed herein and a second
fluorophore with detectably different optical properties, typically
by distinguishing the fluorescence response of the fluorescent
compounds of the invention from that of the second fluorophore.
Where a sample is examined using a flow cytometer, examination of
the sample optionally includes isolation of particles within the
sample based on the fluorescence response by using a sorting
device. In certain embodiments, the illumination source is used to
form a covalent bond between the present pH-sensitive fluorescent
dye compound and an analyte of interest. In this instance the
pH-sensitive fluorescent dye comprises a photoactivatable reactive
group, such as those discussed above.
Kits:
In certain embodiments, kits are provided for determining the pH of
a sample comprising:
(a) one or more of the pH-sensitive fluorescent dye compounds
described herein;
(b) one or more containers; and optionally
(c) instructions for determining the pH of the sample.
In certain embodiments, kits are provided for determining the pH of
a sample comprising:
(a) one or more of the pH-sensitive fluorescent dye compositions
described herein;
(b) one or more containers; and optionally
(c) instructions for determining the pH of the sample.
In certain embodiments, the kits further comprise one or more of
the following: a buffering agent, a purification medium, a vial
comprising the sample, or an organic solvent.
As used herein, the term "kit" refers to a packaged set of related
components, typically one or more pH-sensitive fluorescent dye
compounds or compositions. In certain embodiments, the kits
disclosed herein comprise one or more of the pH-sensitive
fluorescent dye compounds described herein, one or more carriers
suitable for in vitro or in vivo applications, and one or more
containers in which to store the one or more pH-sensitive
fluorescent dyes and/or one or more carriers, such as solvents,
buffers, stabilizers, pH adjusting agents, etc. The kit optionally
contains instructions for how to prepare the one or more
pH-sensitive fluorescent dyes or how to prepare a composition
containing the one or more pH-sensitive fluorescent dye, and how to
administer the dye or composition containing the dye. In certain
embodiments, the kit comprises instructions for performing an assay
that detects the pH or pH changes in samples. In certain
embodiments, the assay is an in vitro assay. In certain
embodiments, the assay is an in vivo assay. The kit may further
comprise one or more pieces of equipment to administer the dye
compound, or composition comprising the pH-sensitive fluorescent
dye compound including, but not limited to, syringes, pipettes,
pipette bulbs, spatulas, vials, syringe needles, and various
combinations thereof.
In certain embodiments, the kits provided herein comprise indicator
solutions or indicator "dipsticks", blotters, culture media,
cuvettes, and the like. In certain embodiments, the kits provided
herein comprise indicator cartridges (where a kit component is
bound to a solid support) for use in an automated detector. In
certain embodiments, the kits provided herein further comprise
molecular weight markers, wherein said markers are selected from
phosphorylated and non-phosphorylated polypeptides, calcium-binding
and non-calcium binding polypeptides, sulfonated and non-sulfonated
polypeptides, and sialylated and non-sialylated polypeptides. In
certain embodiments, the kits provided herein further comprise a
member selected from a fixing solution, a detection reagent, a
standard, a wash solution, and combinations thereof.
Synthesis and Processes of Preparation of the pH-Sensitive
Fluorescent Dye Compounds
In certain embodiments, processes are provided for synthesizing a
compound of structural formula (I):
##STR00015##
the process comprising: contacting a compound of structural formula
(III):
##STR00016## with a compound of structural formula (IV):
##STR00017## to form a compound of structural formula (I),
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are as previously
defined.
An exemplary reaction scheme is shown in detail below:
##STR00018##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, R.sup.10 are as described herein.
In certain embodiments, any one of the above methods of synthesis
further comprises a purifying step. In certain more particular
embodiments, the purifying step comprises at least one of: column
chromatography, trituration, recrystallization, filtration, or
aqueous separation.
A detailed description of the present teachings having been
provided above, the following examples are given for the purpose of
illustrating the invention and shall not be construed as being a
limitation on the scope of the invention or claims.
EXAMPLES
Referring to the examples that follow, pH-sensitive fluorescent dye
compounds disclosed herein were synthesized using the methods
described herein, or other methods, which are known in the art.
It should be understood that the organic compounds according to the
invention may exhibit the phenomenon of tautomerism. As the
chemical structures within this specification can only represent
one of the possible tautomeric forms, it should be understood that
the invention encompasses any tautomeric form of the drawn
structure.
Chemical Synthesis of the Fluorescent pH-Sensitive Dye Compounds
Disclosed Herein
Example 1
Preparation of Compound (3)
##STR00019##
2-(bis(carboxymethyl)amino)-6-(4-((2-(dimethylamino)-4-(6-(dimethylamino)--
3-(dimethyliminio)-3H-xanthen-9-yl)-5-methoxyphenyl)(ethyl)amino)butanamid-
o)hexanoate (Compound 3)
To a solution of Compound (2) (24.0 mg, 0.065 mmol) and
triethylamine (45 .mu.L, 0.325 mmol) in DMSO (1 mL) was added
Compound (1) (40 mg, 0.059 mmol) dissolved in DMF (1 mL) and the
mixture was stirred at room temperature for 18 hours. All volatile
materials were removed by vacuum pump. The crude mixture was
treated with 1 mL of water, and was purified by chromatography on
C-18 (Biotage SNAP 12 g, MeOH:H.sub.2O=0:100.about.30:70) to afford
Compound (3) (38 mg, 81%) as a dark purple powder. H.sup.1 NMR
(MeOD): 7.40 (d, 2H), 7.10 (m, 4H), 6.91 (2H, s), 3.75 (s, 3H),
3.65 (q, 4H), 3.43 (m, 2H), 3.40 (m, 2H), 3.30 (s, 12H), 3.18 (m,
2H), 2.75 (s, 6H), 2.23 (m, 2H), 1.90 (b, 4H), 1.55 (m, 4H), 1.23
(t, 2H,), 1.10 (t, 3H).
Example 2
Preparation of Compound (4)
##STR00020##
N-(9-(4-((8-(2-(acetoxymethoxy)-2-oxoethyl)-9-((acetoxymethoxy)carbonyl)-2-
,6,15-trioxo-3,5-dioxa-8,14-diazaoctadecan-18-yl)(ethyl)amino)-5-(dimethyl-
amino)-2-methoxyphenyl)-6-(dimethylamino)-3H-xanthen-3-ylidene)-N-methylme-
thanaminium (Compound 4)
Compound (3) (38 mg, 0.048 mmol) was dissolved in DMF (2 mL).
Triethylamine (50 .mu.L, 0.336 mmol) was added into the solution.
Bromomethyl acetate (0.05 mL, 0.48 mmol) was added dropwise, the
white smoke was formed during addition. The mixture was stirred at
room temperature for 1 hour. All volatile materials were removed
and the residue was dried by vacuum pump. The resulting residue was
dissolved in chloroform (20 mL) and washed with 1:1 saturated
NaHCO.sub.3-water, the organic layer was dried and concentrated,
purified by chromatography on silica gel column (Biotage, SNAP 25
g, MeOH: chloroform=0-10%) to afford Compound (4) (40 mg, 77%) as a
dark purple solid. H.sup.1 NMR (d6-DMSO) 7.80 (t, 1H), 7.30 (d,
2H), 7.15 (d, 2H), 6.89 (s, 2H), 6.75 (d, 2H), 5.60 (m, 6H), 4.00
(s, 4H), 3.62 (s, 3H), 3.60 (q, 4H), 3.40 (m, 2H), 3.20 (s, 12H),
3.00 (m, 2H), 2.60 (s, 6H), 2.10 (m, 2H), 2.00 (s, 9H), 1.70 (s,
2H), 1.70 (m, 2H), 1.50 (m, 2H), 1.30 (m, 2H), 1.10 (m, 2H), 1.0
(t, 3H). LCMS: 1007.1 (M+1).
Example 3
Preparation of Compounds (7) and (8)
##STR00021##
Compound (6) (9.00 g, 18.3 mmol, see, Wu et al., Org. Lett.
10:1779-1782 (2008), herein incorporated by reference in its
entirety) was treated with DMSO (56 mL) and diallylamine (21.2 g,
219 mmol) at room temperature, and the mixture was heated to
90.degree. C. for 36 hours. The mixture was cooled to room
temperature, and quenched by adding water (250 mL). The product was
extracted with ethyl acetate (200 mL.times.3). The combined organic
layers were washed by water (200 mL.times.2), brine (300 mL), dried
by MgSO.sub.4, and evaporated. The crude product was purified by
chromatography on silica gel column (Biotage, SNAP 340 g,
6.5.times.18 cm, ethyl acetate:hexanes=1:2) to give Compound (7)
(3.30 g, 47%, white solid), and Compound (8) (1.10 g, 20%, pale
yellow solid).
Example 4
Preparation of Compound (9)
##STR00022##
Compound (7) (149 mg, 0.386 mmol) was treated with anhydrous DCM (5
mL) and Tf.sub.2O (80 .mu.L, 0.476 mmol), and the mixture was
stirred at room temperature. After 30 min stirring, Compound (5)
(150 mg, 0.486 mmol) in DCM (2 mL) and DIEA (120 .mu.L, 0.689 mmol)
were added. The reaction was stirred at room temperature for 16
hours. Evaporation gave a crude product. The crude product was
purified by chromatography on silica gel column (Biotage, SNAP 25
g, 2.5.times.8 cm, MeOH:CHCl.sub.3=1:10 1:4) to give Compound (9)
(200 mg, 62%) as a purple gummy material.
Example 5
Preparation of Compounds (14) and (15)
##STR00023##
A solution of 4-methoxyaniline (13) (10.0 g, 81.2 mmol) in DMF (100
mL) was treated with 60% NaH (8.10 g, 203 mmol) and iodoethane
(26.0 mL, 235 mmol). The temperature of the mixture was kept at
0.degree. C. by ice bath in first 3 hour stirring. The ice bath was
removed and the mixture was stirred at room temperature for another
12 hours. Water (200 mL) was added to quench this reaction. The
product was extracted by ethyl acetate (200 mL.times.3). The
combined organic layers were washed by water (200 mL.times.5),
brine (200 mL), and dried over sodium sulfate. Filtration and
evaporation gave Compound (14) (16.2 g, .about.100%) as a dark
green oil.
To a solution of N,N-diethyl-4-methoxyaniline (14) (6.0 g, 27 mmol)
in water (500 mL) and acetic acid (50 mL) was added a solution of
NaNO.sub.2 (3.8 g, 55 mmol) in water (70 mL) dropwise within 30 min
at room temperature. The reaction mixture was stirred at room temp
for 3 hours. The product was extracted by ethyl acetate (150
mL.times.3). The combined organic layers were washed by 1M KOH (100
mL.times.4), brine (100 mL) and dried over anhydrous sodium
sulfate. Filtration and evaporation gave a crude product as dark
red oil. The crude product was purified by chromatography on silica
gel column (Biotage, SNAP 100 g, 3.5.times.16 cm, from 15%
CHCl.sub.3 in hexanes to 100% CHCl.sub.3) to give Compound (15)
(4.90 g, 81%) as a dark red oil.
Example 6
Preparation of Compounds (16) and (17)
##STR00024##
To palladium on activated charcoal (650 mg) was added ethanol (15
mL) under argon carefully. A solution of Compound (15) (5.30 g,
23.6 mmol) in ethanol (75 mL) was treated with the palladium
suspension solution by a pipet. The solution was equipped with a
3-way stopcock attached by a 3 L balloon filled with hydrogen. The
solution was evacuated and refilled with hydrogen carefully three
times. The solution was stirred under hydrogen atmosphere for 18
hours. Filtration through a Celite pad was performed to remove
palladium, and the filtrate was concentrated to dryness to get
crude product. The crude product was purified by chromatography on
silica gel column (Biotage, SNAP 100 g, 3.5.times.16 cm, ethyl
acetate:hexanes=1:4) to give Compound (16) (4.14 g, 90%, as light
brown oil).
A solution of Compound (16) (4.14 g, 21.3 mmol) in DCM (60 mL) was
treated with acetyl chloride (1.80 mL, 25.6 mmol) and triethylamine
(4.44 mL, 32.0 mmol) at 0.degree. C. The mixture was stirred at
0.degree. C. for 30 min, and quenched by adding MeOH (1 mL). The
mixture was diluted by ethyl acetate (300 mL) and the solution was
washed by water (200 mL), brine (150 mL), and dried over anhydrous
sodium sulfate. Evaporation gave pure crude product of Compound
(17) (4.73 g, 94%) as a light brown oil. No further purification
was required.
Example 7
Preparation of Compounds (18) and (19)
##STR00025##
A solution of Compound (17) (4.73 g, 20.0 mmol) in anhydrous THF
(15 mL) was treated with BH.sub.3-THF (60 mL of 1M solution in THF,
60 mmol) carefully. The mixture was heated to reflux for 2 hours
and cooled to room temperature. MeOH (60 mL) was added to the
solution slowly to quench extra amount of borane complex at room
temp, and the mixture was heated to reflux for 10 min. The solution
was cooled to room temperature, and evaporated to dryness. The
residue was dissolved in 300 mL of ethyl acetate, and washed by
saturated sodium bicarbonate (150 mL.times.2), brine (100 mL), and
dried over anhydrous sodium sulfate. Evaporation gave pure crude
product of Compound (18) (4.12 g, 93%) as a light brown oil. No
further purification was required.
A solution of Compound (18) (4.06 g, 18.3 mmol) in 40 mL of DMF was
treated with methyl 4-bromobutanoate (9.23 mL, 73.1 mmol), NaI
(1.36 g, 9.07 mmol), and DIEA (9.44 mL, 54.2 mmol), and the mixture
was heated to 100.degree. C. for 22 hours. The reaction was cooled
to room temperature, 200 mL of water were added, and extracted by
ethyl acetate (150 mL.times.3). The combined organic layers were
washed by water (150 mL.times.3), brine (100 mL), dried over
anhydrous sodium sulfate, and evaporated to give crude product. The
crude product was purified by chromatography on silica gel column
(Biotage, SNAP 100 g, 3.5.times.16 cm, ethyl acetate:hexanes=1:4)
to give Compound (19) (4.60 g, 79%) as light brown oil.
Example 8
Preparation of Compound (20)
##STR00026##
Compound (7) (2.15 g, 5.54 mmol) was treated with anhydrous DCM (50
mL) and Tf.sub.2O (0.94 mL, 0.5.60 mmol), and the mixture was
stirred at room temperature. After 30 min of stirring, Compound
(19) (2.13 g, 6.65 mmol) in DCM (15 mL) and DIEA (1.25 mL, 7.18
mmol) were added. The reaction was stirred at room temperature for
22 hours. Evaporation gave crude product. The crude product was
purified by chromatography on silica gel column (Biotage, SNAP 100
g, 3.5.times.16 cm, H.sub.2O:MeCN:HOAc=1:10:0.1.about.1:6:0.1) to
give Compound (20) (2.58 g, 62%) as a purple gummy material.
Example 9
Preparation of Compound (25)
##STR00027##
A solution of Compound (8) (170 mg, 0.553 mmol) in DMF (5 mL) was
treated with 60% NaH (33.0 mg, 0.825 mmol) and allyl bromide (134
mg, 1.11 mmol) at 0.degree. C. The mixture was allowed warm to room
temperature and stirred overnight (.about.16 hours). The reaction
was quenched by water (20 mL). The solution was extracted by ethyl
acetate (30 mL.times.3). The combined organic layers were washed by
water (30 mL.times.3), brine (50 mL), dried over magnesium sulfate
and evaporated to give Compound (25) (210 mg, .about.100%) as pale
yellow solid. No further purification was required.
Example 10
Preparation of Compound (29)
##STR00028##
Compound (25) (210 mg, 0.605 mmol) was treated with anhydrous DCM
(8 mL) and Tf.sub.2O (121 .mu.L, 0.719 mmol), and the mixture was
stirred at room temperature. After 30 min stirring, Compound (5)
(239 mg, 0.774 mmol) in DCM (6 mL) and DIEA (160 .mu.L, 0.910 mmol)
were added. The reaction was stirred at room temperature for 20
hours. Evaporation gave crude product. The crude product was
purified by chromatography on silica gel column (Biotage, SNAP 50
g, 3.5.times.8.5 cm, MeOH:CHCl.sub.3=1:10 1:4) to give Compound
(29) (412 mg, 87%) as a gray gummy material.
Example 11
Preparation of EGF Conjugate of Compound (4)
0.6 mg of a 5 mg/ml EGF stock solution (120 .mu.L, 0.098 .mu.mol)
was added to a 2 mL vial. 0.6 mg of Compound (4) (0.88 mol) was
dissolved in 100 .mu.L of DMSO, and 23 .mu.L of the solution was
added into the vial with EGF. The dye to protein molar ratio was
.about.2.50 .mu.L of Triethylamine was dissolved in 0.5 mL of DMSO.
6 .mu.L of the triethylamine solution was added to the EGF solution
and stirred, covered, for 24 hours. About 1/10 volume of 1.5 M
hydroxylamine (pH 8.0) was added to stop the reaction and stirred
for about 30 minutes at room temperature. The conjugate was
purified on a P-2F column (15.times.1 cm) with PBS (pH 7.2). 3-6
fractions were collected containing 1-2 mL each and run on a TLC in
CMA (70:25:5) to confirm the absence of free dye and processed by
HPLC to check for unlabeled EGF. Fractions containing free dyes and
fractions containing unlabeled EGF were discarded. Fractions
containing no free dye and no unlabeled EGF were combined as the
final product. The A566 nm/A280 nm was measured and the conjugate
concentration was determined (80 .mu.g/ml) and degree of
substitution (DOS=1) by absorbance. BSA powder was added to the
conjugate solution (final concentration of 1%) and stirred to
gently dissolve. The EGF conjugate was frozen in dry ice for at
least 1 hour and lyophilized for at least 18 hours.
Biological Application Examples of the Fluorescent pH-Sensitive Dye
Compounds
Example 12
Correlation Between pH and Fluorescence of the pH-Sensitive Dye
Compounds Provided Herein
The fluorogenic nature of the pH-sensitive fluorescent dye
compounds described herein makes them very useful for studying a
variety of internalization processes that occur in cells, such as
phagocytosis and endocytosis. This is because upon internalization,
there is a drop in pH inside the phagosome or endosome which
results in an increase of fluorescence from the pH-sensitive
fluorescent dye compounds. Conjugation of the pH-sensitive
fluorescent dye compounds to biomolecules of interest provide for
convenient assays of internalization of these molecules. Examples
include transferrin, EGF and low-density lipoprotein (LDL) for
studying receptor-mediated endocytosis, and labeled bioparticles,
such as E. coli, Staphylococcus, and zymosan for studying
phagocytosis. Assays using these fluorogenic bioconjugates offer
significant advantages over existing techniques due to the fact
that the pH-sensitive fluorescent dye compounds are relatively
non-fluorescent at the neutral pH outside the cell. This reduces or
eliminates the need for wash steps and quencher dyes normally
needed to reduce background signal from bioconjugates outside of
the cells.
The study of the fluorescent response of the pH-sensitive
fluorescent dye compounds to changes in pH was performed in aqueous
buffers (in concentrations around 10 .mu.mol/mL). The results of
the titrations for Compound (1) are shown in FIG. 3.
Example 13
Intracellular Uptake of the pH-Sensitive Fluorescent Dye
Compounds
FIG. 2, Panel B shows cellular internalization of Compound (1).
Cells were imaged using standard fluorescent illumination and
microscopy.
Example 14
Live Cell Phagocytosis/Endocytosis with pH-Sensitive Fluorescent
Dye Conjugates
A 431 cells were grown in complete medium on 35 mm poly-D-lysine
coated glass bottom culture dishes from MatTek. On the day of the
assay, cells were rinsed once with LCIS+1% BSA (Live Cell imaging
solution, Cat# A14291DJ, Life Technologies, Carlsbad, Calif.) and
placed at 37.degree. C. Control dishes received LCIS; EGF
pretreatment dishes received 10 .mu.g/mL unlabeled EGF in LCIS.
Cells were incubated at 37.degree. C. for 30 minutes. Cells were
then cooled to 4.degree. C. on ice for 10 minutes. Compound
(4)-labeled EGF was added to the dishes 1:10 at 5 .mu.g/mL from a
50 .mu.g/mL stock in LCIS. These dishes were incubated on ice for
30 minutes, then washed 2.times. with cold LCIS and allowed to warm
to 37.degree. C. for 60 minutes before imaging with standard TRITC
and FITC filter sets on a DeltaVision Core microscope. Cells
pretreated with unlabeled EGF showed no signal owing to the
occlusion of dye-labeled EGF binding sites and internalization of
EGF receptors by excess unlabeled EGF. Specific signal from
untreated plates was from dye-labeled EGF internalization. All
images were matched for gain and exposure times. FIG. 4 shows
internalization of EGF conjugated to Compound (1). The left panel
shows cells pretreated with EGF and the right panel shows cells
treated with dye-conjugated EGF.
pH-sensitive fluorescent dye compounds were made up in LCIS from 5
and 10 mM DMSO stocks respectively, and loaded for imaging as
follows:
10 mL Loading Buffer, Compound (4):
10 .mu.L Compound (4) from a 5 mM DMSO stock was added to 100 .mu.L
Powerload and mixed. 10 mL LCIS was added to this solution for
final concentration of Compound (4) of 5 .mu.M.
Cells were cultured on 35 mm MatTek glass bottom dishes. For
loading, cells were rinsed 1.times. with LCIS and replaced with
loading buffer described above. Cells were incubated at 37.degree.
C. for 60 minutes, then rinsed 2.times. with LCIS and imaged with
standard TRITC or FITC filter sets. (see FIG. 5).
Cells were cultured and loaded as described above with Compound
(4). This solution was removed from the cells and replaced with
potassium LCIS pH 7.4 standard and imaging experiments begun with
cells under constant perfusion by potassium LCIS pH standards of pH
7.4, 7.2, 6.5, and 6.0. As the external pH was changed, the signal
from the dye inside of the cells changed to reflect the
equilibration of protons through the nigericin pores in the
membrane. (Nigericin and valinomycin added at 10 .mu.M each from
DMSO stock.) Images of Compound (4) AM loaded cells at pH 7.4 and
6.0 are shown in FIG. 6. Functional kinetic responses from
individually selected cells in the sample are shown in FIG. 7 as
the pH was changed from 7.4 to 6.0 and back.
Example 15
Antibody Labeling Using pH-Sensitive Fluorescent Dyes
A freshly prepared 10 mM DMSO solution of Compound (1)-maleimide
was added to an IgG solution (6 mg/mL) in PBS buffer at pH 7.0-7.5
in sufficient amount to give 10-20 moles of Compound (1) for each
IgG molecule. The reaction was allowed to proceed for approximately
2 hours at which time the reaction mixture was poured on to a
pre-packed Sephadex G-25 column. The column was eluted with PBS
buffer to collect purified conjugated IgG. TLC analysis of a small
aliquot (.about.5 .mu.L) of the purified IgG indicated no free dye
in the conjugate solution. The degree of labeling (DOL) was
determined through a typical absorbance reading.
TABLE-US-00004 TABLE 4 Dye-conjugated IgG Compound Moles of dye/IgG
molecule Observed DOL 4 10 2.1 4 20 2.93
Labeled IgGs were collected from the column and checked for purity
via thin layer chromatography, to insure that all free dye was
separated from the labeled IgG. These samples were diluted 1:10 to
final concentrations of 10-100 .mu.g/mL into a series of pH
standard solutions from pH 4 to pH 9 and transferred into a 96 well
microplate and scanned for fluorescence in a Molecular Devices
FlexStation 384 plate reader. As can be seen in FIG. 8, relative
fluorescence units (RFU) on the y-axis demonstrate the pH
activation of fluorescent signal from the IgG conjugates,
increasing signal with decreasing pH. pKa (midpoint) values near
6.8 of the conjugated antibodies track closely with the signal from
the pH-sensitive fluorescent dyes.
Example 16
Labeling of Transferrin with pH Sensing Dyes
All materials are from Life Technologies Corp. (Carlsbad, Calif.)
unless otherwise stated. Dissolve transferrin from human serum
(Sigma, T4132) in 0.1 M NaHCO.sub.3, pH 8.3, to a concentration of
10 mg/mL. Make a 10 mg/mL solution of the succinimidyl ester of the
pH-sensitive fluorescent dye compound in dry DMSO and sonicate
briefly to aid in dissolution of the dye. Add a 10 to 30-fold molar
excess of the reactive dye solution to the transferrin solution
dropwise while stirring. Note that the volume of dye added depends
on the specific dye and the amount of transferrin to be labeled.
Protect the reaction vessel from light and stir for .about.1 hour
at room temperature. Purify the conjugate on a P-30M gel filtration
column (BioRad, 150-4150) in PBS, pH 7.2. Centrifuge the conjugate
at 19,000 rpm for 20 minutes to remove aggregates, if present.
Determine the degree of labeling by measuring A560 nm/A280 nm.
Example 17
Monitoring Cytosolic Acidification Associated with Ion Channel or
Transporter Activation
A cytosolically localized version of the pH-sensitive fluorescent
dye compound is be a useful indication of proton influx through ion
channels or transporters. This may be used for screening of
antagonists, agonists, and other modulators of channel/transporter
function.
Example 18
Receptor Internalization Assay
The .beta.-2-Adrenergic Receptor (.beta.2AR) is modified to
incorporate an epitope tag (VSV-G tag) at the N-terminus. A clonal,
stable HEK 293 cell line is established which expresses this
receptor (approximately 1.8 pmol/mg cell homogenate). Anti-VSV-G
antibody labeled with a pH-sensitive fluorescent dye compound
described herein is used to monitor agonist-mediated receptor
internalization in these live cells. The assay is performed in the
presence and absence of a specific agonist, isoproterenol.
a) Isoproterenol-Induced Receptor Internalization in VSV-G-B2
Adrenergic Cells.
For HEK 293 cells it is preferable to coat plates with
poly-D-lysine (Sigma P-6407, 5 mg in 50 ml sterile PBS) prior to
seeding the cells. 30-80 .mu.l/well is added and maintained at room
temperature for 45 minutes. The coating solution is then aspirated,
wash 4.times. (or more) with 100 .mu.l sterile PBS. Plates can be
treated in advance and stored at 4.degree. C. for up to a week
(with the final PBS wash still in the wells). Cultured cells can be
seeded directly into the wells without first drying the plates.
Cultured cells are diluted to .about.1.6.times.10.sup.5 cells/ml in
complete MEM media (Sigma M2279) containing 200 .mu.g/ml G418. 100
.mu.l of cell suspension is pipetted into each assay well of a
poly-D-lysine treated 96-well Packard Viewplate (cell density=16000
cells per well). Plates are then incubated 24-48 hours at
37.degree. C. with 5% CO.sub.2. 250 .mu.g lyophilized pH-sensitive
fluorescent dye compound labeled anti-VSV-G antibody (PA45407) is
reconstituted with 0.5 ml sterile deionized water and mixed
thoroughly (stock concentration 0.5 mg/ml). The mixture is
centrifuged to remove any precipitate. The compound labeled
anti-VSV-G antibody is further diluted to a concentration of 2.5-5
.mu.g/ml using serum-free, phenol red free MEM media. Hoechst 33342
nuclear stain can be added to the 2.5-5 .mu.g/ml antibody solution
to a final concentration of 5 .mu.M. Media is subsequently removed
from the cells and 100 .mu.l antibody and Hoechst solution is added
to each well. The solution is then incubated at room temperature
for 15 minutes. 3 .mu.M working dilution of isoproterenol agonist
(from 10 mM stock in sterile water; Sigma I5627) is added to the
solution and then 50 .mu.l is added to required wells, giving 1
.mu.M final concentration. The wells are incubated at 37.degree. C.
for 30 minutes (in a CO.sub.2 incubator or on the IN Cell Analyzer
3000). The cells are imaged on an IN Cell Analyzer 3000, IN Cell
Analyzer 1000 or a confocal microscope.
b) Internalization of Dye Compound-Labeled Anti-VSV-G Antibody.
HEK 293 cells expressing a VSV-G-.beta.-Adrenergic Receptor are
preincubated with anti-VSV-G antibody-dye compound conjugate and
stimulated with 1 .mu.M isoproterenol. The cells are imaged using
an IN Cell Analyzer 1000. Quantification of the agonist-mediated
response is achieved using a granularity algorithm, which defines
grains as distinct focal regions within a cell that have pronounced
intensity differences from the region of the cell immediately
surrounding the grains. The operator can adjust a variety of
parameters to control what size and intensity of grain will be
counted and analyzed.
c) Internalization of Dye Compound-Labeled Anti-VSV-G Antibody.
HEK VSV-G-.beta.2-Adrenergic Receptor cells are preincubated with
pH-sensitive fluorescent dye compound labeled anti-VSV-G antibody
and increasing concentrations of isoproterenol (0-1 .mu.M) are then
added to the cells. After 30 minutes at 37.degree. C., agonist
mediated internalization is analyzed by measuring the increase in
pH-sensitive fluorescent dye compound fluorescence using an IN Cell
Analyzer 1000 and the granularity analysis algorithm.
Example 19
Detection of Neuronal Cells with a pH-Sensitive Fluorescent Dye
Compound
Astroglial feeder layers are established for one week in culture on
glass bottomed culture dishes, 35 mm diameter, coated with
Poly-L-Lysine. Neurons from embryonic day 18 rat hippocampi are
dissociated in culture medium, and seeded onto the feeder layers at
a density of 25-35,000 cells per mL, 2 mL per dish, and allowed to
grow in neuronal culture medium plus mitotic inhibitors to prevent
glial proliferation.
Cells are pre-stained for 15 minutes with 200 ng/mL Hoescht to
visualize DNA in the nuclei, and 50 ng/mL calcien AM ester to
visualize the cytoplasm by adding 1000.times.DMSO stocks of these
compounds to the cells in complete medium, and then returning them
to the cell culture incubator for 15 minutes at 37.degree. C. Cells
are removed, and the medium is gently poured off. The cells are
immediately placed in 5 .mu.M pH-sensitive fluorescent dye
compound, diluted from a 1 mM DMSO stock into normal saline plus 20
mM HEPES and 20 mM glucose, final pH set to 7.4 with NaOH. The
cells are incubated in labeling solution for ten minutes at room
temperature, and then gently washed twice with saline (above) minus
dye for imaging.
Example 20
Phagocytosis of .beta.1 Amyloid Conjugates
1 mg of beta amyloid 1-42 is labeled with a pH-sensitive
fluorescent dye compound to yield a dye-beta amyloid conjugate,
which is purified by gel filtration to yield a solution of
approximately 200 ng/mL with a degree of labeling between 1 and 2
dye molecules per beta amyloid molecule.
2 mL of J774A.1 cells are seeded onto 35 mm, poly-D-lysine coated
glass bottom culture dishes at a density of 35,000 cells per mL one
day in advance of the study, in serum-free OptiMem culture medium.
The dye-beta amyloid conjugate is filtered through a 0.2 micron
syringe filter, and 20 microliters of the solution is added to the
cells. The culture is returned to the incubator (37.degree. C., 5%
CO.sub.2) for overnight incubation, and imaged on the following
day.
Example 37
Copper-Less Click Reactions Using DIBO-Containing pH-Sensitive
Fluorescent Dye Compounds
a) Preparation of DIBO-Labeled Dye Compounds:
A solution of pH-sensitive dye compound STP ester (10 mg, 0.0134
mmol) and DIBO amine (Cat. No. C10411, Molecular Probes, Carlsbad,
Calif.) (5.0 mg 0.0156 mmol) in DMF (1 mL) is treated with 10 .mu.l
of triethylamine (0.072 mmol) and the mixture is stirred at room
temperature for 1 hour. All volatile materials are removed by
vacuum pump overnight. The crude reaction mixture is dried
carefully by vacuum pump and purified by chromatography on a Silica
gel column (Biotage, SNAP 10 g MeOH:CHCl.sub.3=1:10-1:6) to give a
DIBO-dye conjugate.
a) Live Cell Labeling:
Mammalian cells are grown in an appropriate medium at 37.degree. C.
in 5% CO.sub.2. Supplement the growth medium with an
azide-derivatized metabolite (e.g., Click-iT.RTM. ManNAz, Life
Technologies, Catalog No. C33366) and grow the cells for 2 to 3
days. Wash the cells two times with D-PBS (Life Technologies,
Catalog No. 14190-144) containing 1% fetal bovine serum (FBS).
Label the azide-modified macromolecules at room temperature in the
dark for 1 hour with about 5 to 30 .mu.m DIBO-containing
pH-sensitive dye compound in D-PBS containing 1% FBS. Wash the
cells four times with D-PBS containing 1% FB S. Fix the cells with
4% formaldehyde in D-PBS for 15 minutes at room temperature. Wash
the cells with D-PBS. Optionally, counterstain the cells with an
appropriate counterstain, such as Hoechst 33342 and wash the cells.
Image the cells.
b) Protein Labeling:
Introduce azide into proteins, e.g., using GalNAz in antibodies
using the Click-iT.RTM. O-GlcNAc Enzymatic Labeling System (Life
Technologies, Catalog No. C33368). Modify the protein-bound azide
with DIBO-containing pH-sensitive dye compound. Incubate the
protein in TBS with about 5 to 10 .mu.m DIBO-containing
pH-sensitive dye compound for at least 1 hour at room temperature.
Remove the excess label. Analyze the modified protein.
* * * * *